Phosphorus-containing prodrugs

ABSTRACT

Novel cyclic phosphoramidate prodrugs of drugs of formula I 
                         
their use in delivery of drugs to the liver, their use in enhancing oral bioavailability, and their method of preparation are described.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.11/779,689 filed Jul. 18, 2007, now allowed, which is a continuationapplication of U.S. application Ser. No. 11/601,843 filed Nov. 20, 2006,now abandoned, which is a divisional application of U.S. applicationSer. No. 09/518,501 filed Mar. 3, 2000, now patented, which claimspriority to U.S. Provisional Application Ser. No. 60/153,127, filed Sep.8, 1999, and Provisional Application Ser. No. 60/123,013, filed Mar. 5,1999, and each of which is incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention is directed towards novel prodrugs that generatephosph(on)ate compounds which are biologically active or are furtherphosphorylated to produce biologically active compounds, to theirpreparation, to their synthetic intermediates, and to their uses. Morespecifically, the invention relates to the area of substituted cyclic1,3-propanyl esters wherein the cyclic moiety contains at least oneamino attached to the phosphorus.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedto aid in understanding the invention, but is not admitted to be, or todescribe, prior art to the invention. All cited publications areincorporated by reference in their entirety.

Free phosphorus and phosphonic acids and their salts are highly chargedat physiological pH and therefore frequently exhibit poor oralbioavailability, poor cell penetration and limited tissue distribution(e.g. CNS). In addition, these acids are also commonly associated withseveral other properties that hinder their use as drugs, including shortplasma half-life due to rapid renal clearance, as well as toxicities(e.g. renal, gastrointestinal, etc.) (e.g. Antimicrob Agents Chemother1998 May; 42(5): 1146-50). Phosphates have an additional limitation inthat they are not stable in plasma as well as most tissues since theyundergo rapid hydrolysis via the action of phosphatases (e.g. alkalinephosphatase, nucleotidases).

Prodrugs of phosphorus-containing compounds have been sought primarilyto improve the limited oral absorption and poor cell penetration. Incontrast to carboxylic acid proesters, many phosphonate and phosphateesters fail to hydrolyze in vivo, including simple alkyl esters. Themost commonly used prodrug class is the acyloxyalkyl ester, which wasfirst applied to phosphate and phosphonate compounds in 1983 by Farquharet al., J. Pharm. Sci. 72(3):324 (1983).

Cyclic phosphonate and phosphate esters have also been described forphosphorus-containing compounds. In some cases, these compounds havebeen investigated as potential phosphate or phosphonate prodrugs.Hunston et al., J. Med. Chem. 27: 440-444 (1984). The numbering forthese cyclic esters is shown below:

The cyclic 2′,2′-difluoro-1′,3′-propane ester is reported to behydrolytically unstable with rapid generation of the ring-openedmonoester. Starrett et al. J. Med. Chem. 37: 1857-1864 (1994).

Cyclic 3′,5′-phosphate esters of araA, araC and thioinosine have beensynthesized. Meier et al., J. Med. Chem. 22: 811-815 (1979). Thesecompounds are ring-opened through the action of phosphodiesterases whichusually require one negative charge.

Cyclic 1′,3′-propanyl phosphonate and phosphate esters are reportedcontaining a fused aryl ring, i.e. the cyclosaligenyl ester, Meier etal., Bioorg. Med. Chem. Lett. 7: 99-104 (1997). These prodrugs arereported to generate the phosphate by a “controlled, non-enzymaticmechanism[s] at physiological pH according to the designedtandem-reaction in two coupled steps”. The strategy was purportedly usedto deliver d4-T monophosphate to CEM cells and CEM cells deficient inthymidine kinase infected with HIV-1 and HIV-2.

Unsubstituted cyclic 1′,3′-propanyl esters of the monophosphates of5-fluoro-2′-deoxy-uridine (Farquhar et al., J. Med. Chem. 26: 1153(1983)) and ara-A (Farquhar et al., J. Med. Chem. 28: 1358 (1985)) wereprepared but showed no in vivo activity. In addition, cyclic1′,3′-propanyl esters substituted with a pivaloyloxy methyloxy group atC-1′ was prepared for 5-fluoro-2′-deoxy-uridine monophosphate (5-FdUMP;(Freed et al., Biochem. Pharmac. 38: 3193 (1989); and postulated aspotentially useful prodrugs by others (Biller et al., U.S. Pat. No.5,157,027). In cells, the acyl group of these prodrugs underwentcleavage by esterases to generate an unstable hydroxyl intermediatewhich rapidly broke down to the free phosphate and acrolein following aβ-elimination reaction as well as formaldehyde and pivalic acid.

Unsubstituted cyclic phosphoramidate esters, i.e. cyclic phosphonate andphosphate esters wherein one of the ring oxygens is replaced with an NR,are also known. For example, cyclophosphamide (CPA) is representative ofa class of mustard oncolytics that utilize this prodrug moiety. AlthoughCPA is activated primarily in the liver via a cytochrome P450-catalyzedoxidation, its biological activity is outside the liver. CPA is aneffective immunosuppressive agent as well as an oncolytic agent forextrahepatic cancers because one or more of the intermediate metabolitesproduced following P450 activation diffuses out of the liver and intothe circulation. With time the intermediate(s) enter extrahepatictissues and are thought to undergo a β-elimination reaction to generateacrolein and the active phosphoramide mustard. Both products arereported to be cytotoxic to cells. The mustard cytotoxicity results fromalkylation of DNA (or RNA). Acrolein is reported to exert its toxicityvia several mechanisms, including depletion of glutathione, alkylationof DNA and proteins via a Michael reaction. In addition, acroleinproduces other toxicities such as the dose-limiting bladder toxicitycommonly observed with cyclophosphamide therapy. Since the toxicity ofthese agents often hampers their use as chemotherapy agents, numerousstrategies are under investigation that are designed to enhance P450activity in or near tumors and thereby localize the activation andantiproliferative effect of these agents to the tumor. One strategy usesretroviruses or other well known techniques for introducing genes intotarget tissues (e.g. Jounaidi et al., Cancer Research 58, 4391 (1998)).Other strategies include the placement of encapsulated cells engineeredto produce cytochrome P450s (e.g. Lohr et al., Gene Therapy 5, 1070(1998)) at or near the tumor.

Unsubstituted cyclic phosphoramidate esters have also been prepared aspotential prodrugs of the nucleosides araA and 5-fluoro-2′-deoxyuridine(Farquhar et al., J. Med. Chem. 28, 1358 1361 (1985); J. Med. Chem. 26,1153-1158 (1983)). The compounds were studied in a mouse model ofleukemia where it was assumed that if the prodrug transformation wassimilar to cyclophosphamide, then these agents would be useful fortreating a variety of cancers including leukemias as well as carcinomasof the colon, breast and ovary. In addition, since some of themechanisms that account for tumor cell drug resistance entail a decreasein the enzymes used to synthesize the monophosphate, the strategy wasexpected to possibly be beneficial in treating drug resistant tumors.The compounds were only “marginally effective” in prolonging life spanin the mouse model.

A variety of substituted 1′,3′ propanyl cyclic phosphoramidates, wherein1′ represents the carbon alpha to the nitrogen were prepared ascyclophosphamide analogs (Zon, Progress in Med. Chem. 19, 1205 (1982)).For example, a number of 2′- and 3′-substituted proesters were preparedin order to decrease the propensity of the α,β-unsubstituted carbonylby-product to undergo a Michael reaction. 2′-Substituents includedmethyl, dimethyl, bromo, trifluoromethyl, chloro, hydroxy, and methoxywhereas a variety of groups were used at the 3′-position includingphenyl, methyl, trifluoromethyl, ethyl, propyl, i-propyl, andcyclohexyl. Analogs with a 3′-aryl group e.g.trans-4-phenylcyclophosphamide were “moderately effective in L1210 testsystem and showed no activity in vivo” G. Zu Prog. Med. Chem. 19:205-246 (1982). A variety of 1′-substituted analogs were also prepared.In general these compounds were designed to be “pre-activated”cyclophosphamide analogs that bypass the oxidation step by alreadyexisting as a 1′-substituted analog capable of producing the finalcompound, e.g. hydroperoxide and 1-thioether. A series of 1′-arylanalogs were also prepared in order to enhance the oxidation potential.In contrast to the 1′-hydroperoxy analogs, the 1′-aryl compoundsexhibited either no activity or very poor activity in the standardanticancer in vivo screen assay, i.e. the mouse L1210 assay. The lack ofactivity was postulated to arise from the steric hindrance of the phenyland therefore limited oxidation of the prodrug. Support for thispostulate was the potent activity of the acyclic phenyl keto analogwhich exhibited activity similar to cyclophosphamide. Luderman et al. J.Med. Chem. 29: 716 (1986).

Cyclic esters of phosphorus-containing compounds are reported in thechemical literature, however they were not tested as prodrugs inbiological systems. These cyclic esters include:

-   [1] di and tri esters of phosphoric acids as reported in Nifantyev    et al., Phosphorus, Sulfur Silicon and Related Elements, 113: 1    (1996); Wijnberg et al., EP-180276 A1;-   [2] phosphorus (III) acid esters. Kryuchkov et al., Izv. Akad. Nauk    SSSR, Ser. Khim. 6:1244 (1987). Some of the compounds were claimed    to be useful for the asymmetric synthesis of L-Dopa precursors.    Sylvain et al., DE3512781 A1;-   [3] phosphoramidates. Shih et al., Bull. Inst. Chem. Acad. Sin, 41:    9 (1994); Edmundson et al., J. Chem. Res. Synop. 5: 122 (1989); and-   [4] phosphonates. Neidlein et al., Heterocycles 35:1185 (1993).

Numerous phosphorus-containing compounds are known to exhibitpharmacological activity but remain far from optimal due to one or moreof the above-described limitations. Some of the activities describedinclude phosphonic acids that are useful as antihypertensives andtherapy for heart failure via inhibition of NEP 24.11, phosphonic acidsthat are useful for treating a variety of CNS conditions (stroke,epilepsy, brain and spinal cord trauma, etc.) via binding to excitoryamino acid receptors (e.g. NMDA receptor), bisphosphonic acids that areuseful for treating osteoporosis, phosphonic acids that are useful aslipid lowering agents (e.g. squalene synthase inhibitors), phosphonatesthat are useful in treating inflammation (e.g. collagenase inhibitors),phosphonates and phosphates that are useful in treating diabetes, cancerand parasitic and viral infections.

Phosphates and phosphonates that are known to be particularly useful inglucose lowering activity and therefore are anticipated to be useful intreating diabetes are compounds that bind to the AMP site of fructose1,6-bisphosphatase (FBPase) as described in U.S. Pat. No. 5,658,889, WO98/39344, WO 98/39343, and WO 98/39342. Other examples ofphosphorus-containing drugs include squalene synthetase inhibitor (e.g.BMS 188494).

A large class of drugs known to be active against hepatitis aregenerally nucleoside or nucleotide analogs that are phosphorylatedinside cells to produce the biologically active triphosphate. Examplesinclude Lamivudine (3TC) and Vidarabine (araA). In each case, the druginterferes with viral replication via the triphosphate form througheither inhibition of the viral DNA polymerases or DNA chain termination.Some specificity for virus-infected cells is gained by both preferentialphosphorylation of the drug by virally-encoded kinases as well as byspecific inhibition of viral DNA polymerases. Nevertheless, many of thenucleoside-based drugs are associated with significant non-hepatictoxicity. For example, araA frequently produces neurological toxicity(40%) with many patients showing myalgia or a sensory neuropathy withdistressing pain and abnormalities in nerve conduction and a few showingtremor, dysarthria, confusion or even coma. Lok et al., J Antimicrob.Chemotherap. 14: 93-99 (1984). In other cases, the efficacy and/ortherapeutic index of nucleosides is compromised by poor phosphorylationefficiencies and therefore low levels of the biologically activetriphosphate (e.g. Yamanaka et al., Antimicrob. Agents and Chemother.43, 190 (1999)).

Phosphonic acids also show antiviral activity. In some cases thecompounds are antivirals themselves (e.g. phosphonoformic acid), whereasin other cases they require phosphorylation to the disphosphate, e.g.9-(2-phosphonylmethoxyethyl)adenine (PMEA, Adefovir). Frequently, thesecompounds are reported to exhibit enhanced activity due to either poorsubstrate activity of the corresponding nucleoside with viral kinases orbecause the viral nucleoside kinase which is required to convert thenucleoside to the monophosphate is down regulated viral resistance.Monophosphates and phosphonic acids, however, are difficult to deliverto virally-infected cells after oral administration due to their highcharge and in the case of the monophosphate instability in plasma. Inaddition, these compounds often have short half-lives (e.g. PMEA,Adefovir) due in most cases to high renal clearance. In some cases, thehigh renal clearance can lead to nephrotoxicities or be a majorlimitation in diseases such as diabetes where renal function is oftencompromised.

Liver cancer is poorly treated with current therapies. In general, livertumors are resistant to radiotherapy, respond poorly to chemotherapy andare characterized by a high degree of cell heterogeneity. Oncolyticnucleosides such as 5-fluoro-2′-deoxyuridine, have also shown a poorresponse against primary liver cancers.

SUMMARY OF THE INVENTION

The present invention is directed towards novel prodrugs that generatephosph(on)ate compounds, their preparation, their syntheticintermediates, and their uses. In one aspect, the invention is directedtowards the use of the prodrugs to enhance oral drug delivery. Anotheraspect of the invention is directed to the use of the prodrugs toenhance the level of the biologically active drug in the liver. Anotheraspect of the invention is the use of the prodrugs to treat diseasesthat benefit from enhanced drug distribution or specificity to the liverand like tissues and cells, including hepatitis, cancer, liver fibrosis,malaria, other viral and parasitic infections, and metabolic diseaseswhere the liver is responsible for the overproduction of the biochemicalend product, e.g. glucose (diabetes); cholesterol, fatty acids andtriglycerides (hyperlipidemia) (atherosclerosis) (obesity). In anotheraspect, the prodrugs are used to prolong pharmacodynamic half-life ofthe drug. In addition, the prodrug methodology of the current inventionis used to achieve sustained delivery of the parent drug. In anotheraspect, the prodrugs are used to increase the therapeutic index of thedrug. Another aspect of the invention is the use of the prodrugs incombination with techniques that elevate P450 activity in specifictissues. In another aspect of the invention, a method of making theseprodrugs is described. A further aspect is the novel intermediates tothese prodrugs. In another aspect, the prodrugs are also useful in thedelivery of diagnostic imaging agents to the liver.

One aspect of the present invention concerns compounds that areconverted in vitro or in vivo to the corresponding M-PO₃ ²—, MP₂O₆ ³—,MP₃O₉ ⁴—, and MP(O)(NHR⁶)O⁻ and are of formula

wherein:

V, W, and W′ are independently selected from the group consisting of —H,alkyl, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl,substituted heteroaryl, 1-alkenyl, and 1-alkynyl; or

together V and Z are connected via an additional 3-5 atoms to form acyclic group containing 5-7 ring atoms, optionally 1 heteroatom,substituted with hydroxy, acyloxy, alkoxycarbonyloxy, oraryloxycarbonyloxy attached to a carbon atom that is three atoms fromboth Y groups attached to the phosphorus; or

together V and Z are connected via an additional 3-5 atoms to form acyclic group, optionally containing 1 heteroatom, said cyclic group isfused to an aryl group at the beta and gamma position to the Y adjacentto V;

together V and W are connected via an additional 3 carbon atoms to forman optionally substituted cyclic group containing 6 carbon atoms andsubstituted with one substituent selected from the group consisting ofhydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, andaryloxycarbonyloxy, attached to one of said additional carbon atoms thatis three atoms from a Y attached to the phosphorus;

together Z and W are connected via an additional 3-5 atoms to form acyclic group, optionally containing one heteroatom, and V must be aryl,substituted aryl, heteroaryl, or substituted heteroaryl;

together W and W′ are connected via an additional 2-5 atoms to form acyclic group, optionally containing 0-2 heteroatoms, and V must be aryl,substituted aryl, heteroaryl, or substituted heteroaryl;

Z is selected from the group consisting of —CHR²OH, —CHR²OC(O)R³,—CHR²OC(S)R³, —CHR²OC(S)OR³, —CHR²OC(O)SR³, —CHR²OCO₂R³, OR², —SR²,—CHR²N₃, —CH₂aryl, —CH(aryl)OH, —CH(CH═CR² ₂)OH, —CH(C≡CR²)OH, R², —NR²₂, —OCOR³, —OCO₂R³, —SCOR³, —SCO₂R³, —NHCOR², —NHCO₂R³, —CH₂NHaryl,—(CH₂)_(p-)OR¹², and —(CH₂)_(p-)SR¹²;

p is an integer 2 or 3;

with the provisos that:

a) V, Z, W, W′ are not all —H; and

b) when Z is —R², then at least one of V, W, and W′ is not —H, alkyl,aralkyl, or alicyclic;

R² is selected from the group consisting of R³ and —H;

R₃ is selected from the group consisting of alkyl, aryl, alicyclic, andaralkyl;

R⁶ is selected from the group consisting of —H, lower alkyl,acyloxyalkyl, alkoxycarbonyloxyalkyl, and lower acyl;

R¹² is selected from the group consisting of —H, and lower acyl;

each Y is independently selected from the group consisting of —O—, —NR⁶—with the proviso that at least one Y is —NR⁶—;

M is selected from the group that attached to PO₃ ²⁻, P₂O₆ ³, P₃O₉ ⁴⁻ orP(O)(NHR⁶)O⁻ is a biologically active agent, but is not an FBPaseinhibitor, and is attached to the phosphorus in formula I via a carbon,oxygen, sulfur or nitrogen atom;

with the provisos that:

1) M is not —NH(lower alkyl), —N(lower alkyl)₂, —NH(lower alkylhalide),—N(lower alkylhalide)₂, or —N(lower alkyl) (lower alkylhalide); and

2) R⁶ is not lower alkylhalide;

and pharmaceutically acceptable prodrugs and salts thereof.

The present invention provides several novel methods of making theprodrugs of the present invention. One method relies on the reaction ofthe following novel P(III) reagent:

The resulting phosphite is then oxidized to the cyclic phosphoramidate.

A second method relies on the reaction of a novel P(V) reagent:

A third method relies on reacting another novel P(V) compound with adiamine or amino alcohol:

Since these compounds have asymmetric centers, the present invention isdirected not only to racemic and diastereomeric mixtures of thesecompounds, but also to individual stereoisomers. The present inventionalso includes pharmaceutically acceptable and/or useful salts of thecompounds of formula I, including acid addition salts. The presentinventions also encompass prodrugs of compounds of formula I.

DEFINITIONS

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

The term “aryl” refers to aromatic groups which have 5-14 ring atoms andat least one ring having a conjugated pi electron system and includescarbocyclic aryl, heterocyclic aryl and biaryl groups, all of which maybe optionally substituted. Suitable aryl groups include phenyl andfuran-2,5-diyl.

Carbocyclic aryl groups are groups wherein the ring atoms on thearomatic ring are carbon atoms. Carbocyclic aryl groups includemonocyclic carbocyclic aryl groups and polycyclic or fused compoundssuch as optionally substituted naphthyl groups.

Heterocyclic aryl or heteroaryl groups are groups having from 1 to 4heteroatoms as ring atoms in the aromatic ring and the remainder of thering atoms being carbon atoms. Suitable heteroatoms include oxygen,sulfur, and nitrogen. Suitable heteroaryl groups include furanyl,thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolyl, pyridyl-N-oxide,pyrimidyl, pyrazinyl, imidazolyl, and the like, all optionallysubstituted.

The term “biaryl” represents aryl groups containing more than onearomatic ring including both fused ring systems and aryl groupssubstituted with other aryl groups. Such groups may be optionallysubstituted. Suitable biaryl groups include naphthyl and biphenyl.

The term “alicyclic” means compounds which combine the properties ofaliphatic and cyclic compounds. Such cyclic compounds include but arenot limited to, aromatic, cycloalkyl and bridged cycloalkyl compounds.The cyclic compound includes heterocycles. Cyclohexenylethyl andcyclohexylethyl are suitable alicyclic groups. Such groups may beoptionally substituted.

The term “optionally substituted” or “substituted” includes groupssubstituted by one to four substituents, independently selected fromlower alkyl, lower aryl, lower aralkyl, lower alicyclic, hydroxy, loweralkoxy, lower aryloxy, perhaloalkoxy, aralkoxy, heteroaryl,heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, amino, guanidino,amidino, halo, lower alkylthio, oxo, acylalkyl, carboxy esters,carboxyl, -carboxamido, nitro, acyloxy, aminoalkyl, alkylaminoaryl,alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino,phosphono, sulfonyl, -carboxamidoalkylaryl, -carboxamidoaryl,hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy-,aminocarboxamidoalkyl-, cyano, lower alkoxyalkyl, lower perhaloalkyl,and arylalkyloxyalkyl. “Substituted aryl” and “substituted heteroaryl”preferably refers to aryl and heteroaryl groups substituted with 1-3substituents. Preferably these substituents are selected from the groupconsisting of lower alkyl, lower alkoxy, lower perhaloalkyl, halo,hydroxy, and amino.

The term “-aralkyl” refers to an alkylene group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,and may be optionally substituted. “Heteroarylalkyl” refers to analkylene group substituted with a heteroaryl group.

The term “-alkylaryl” refers to an aryl group substituted with an alkylgroup. “Lower-alkylaryl” refers to such groups where alkyl is loweralkyl.

The term “lower” referred to herein in connection with organic radicalsor compounds respectively defines such as with up to and including 10,preferably up to and including 6, and advantageously one to four carbonatoms. Such groups may be straight chain, branched, or cyclic.

The terms “arylamino” (a), and “aralkylamino” (b), respectively, referto the group —NRR′ wherein respectively, (a) R is aryl and R′ ishydrogen, alkyl, aralkyl or aryl, and (b) R is aralkyl and R′ ishydrogen or aralkyl, aryl, alkyl.

The term “acyl” refers to —C(O)R where R is alkyl and aryl.

The term “carboxy esters” refers to —C(O)OR where R is alkyl, aryl,aralkyl, and alicyclic, all optionally substituted.

The term “carboxyl” refers to —C(O)OH.

The term “oxo” refers to ═O in an alkyl group.

The term “amino” refers to —NRR′ where R and R′ are independentlyselected from hydrogen, alkyl, aryl, aralkyl and alicyclic, all except Hare optionally substituted; and R and R¹ can form a cyclic ring system.

The term “-carboxylamido” refers to —CONR₂ where each R is independentlyhydrogen or alkyl.

The term “halogen” or “halo” refers to —F, —Cl, —Br and —I.

The term “alkylaminoalkylcarboxy” refers to the groupalkyl-NR-alk-C(O)—O— where “alk” is an alkylene group, and R is a H orlower alkyl.

The term “alkyl” refers to saturated aliphatic groups includingstraight-chain, branched chain and cyclic groups. Alkyl groups may beoptionally substituted. Suitable alkyl groups include methyl, isopropyl,and cyclopropyl.

The term “cyclic alkyl” or “cycloalkyl” refers to alkyl groups that arecyclic of 3 to 10 atoms, more preferably 3 to 6 atoms. Suitable cyclicgroups include norbornyl and cyclopropyl. Such groups may besubstituted.

The term “heterocyclic” and “heterocyclic alkyl” refer to cyclic groupsof 3 to 10 atoms, more preferably 3 to 6 atoms, containing at least oneheteroatom, preferably 1 to 3 heteroatoms. Suitable heteroatoms includeoxygen, sulfur, and nitrogen. Heterocyclic groups may be attachedthrough a nitrogen or through a carbon atom in the ring. Theheterocyclic alkyl groups include unsaturated cyclic, fused cyclic andspirocyclic groups. Suitable heterocyclic groups include pyrrolidinyl,morpholino, morpholinoethyl, and pyridyl.

The term “phosphono” refers to —PO₃R₂, where R is selected from thegroup consisting of —H, alkyl, aryl, aralkyl, and alicyclic.

The term “sulphonyl” or “sulfonyl” refers to —SO₃R, where R is H, alkyl,aryl, aralkyl, and alicyclic.

The term “alkenyl” refers to unsaturated groups which contain at leastone carbon-carbon double bond and includes straight-chain,branched-chain and cyclic groups. Alkenyl groups may be optionallysubstituted. Suitable alkenyl groups include allyl. “1-alkenyl” refersto alkenyl groups where the double bond is between the first and secondcarbon atom. If the 1-alkenyl group is attached to another group, e.g.it is a W substituent attached to the cyclic phosphoramidate, it isattached at the first carbon.

The term “alkynyl” refers to unsaturated groups which contain at leastone carbon-carbon triple bond and includes straight-chain,branched-chain and cyclic groups. Alkynyl groups may be optionallysubstituted. Suitable alkynyl groups include ethynyl. “1-alkynyl” refersto alkynyl groups where the triple bond is between the first and secondcarbon atom. If the 1-alkynyl group is attached to another group, e.g.it is a W substituent attached to the cyclic phosphoramidate, it isattached at the first carbon.

The term “alkylene” refers to a divalent straight chain, branched chainor cyclic saturated aliphatic group.

The term “acyloxy” refers to the ester group —O—C(O)R, where R is H,alkyl, alkenyl, alkynyl, aryl, aralkyl, or alicyclic.

The term “aminoalkyl-” refers to the group NR₂-alk- wherein “alk” is analkylene group and R is selected from H, alkyl, aryl, aralkyl, andalicyclic.

The term “alkylaminoalkyl-” refers to the group alkyl-NR-alk- whereineach “alk” is an independently selected alkylene, and R is H or loweralkyl. “Lower alkylaminoalkyl-” refers to groups where each alkylenegroup is lower alkylene.

The term “arylaminoalkyl-” refers to the group aryl-NR-alk- wherein“alk” is an alkylene group and R is H, alkyl, aryl, aralkyl, andalicyclic. In “lower arylaminoalkyl-”, the alkylene group is loweralkylene.

The term “alkylaminoaryl-” refers to the group alkyl-NR-aryl- wherein“aryl” is a divalent group and R is H, alkyl, aralkyl, and alicyclic. In“lower alkylaminoaryl-”, the alkylene group is lower alkyl.

The term “alkoxyaryl-” refers to an aryl group substituted with analkyloxy group. In “lower alkyloxyaryl-”, the alkyl group is loweralkyl.

The term “aryloxyalkyl-” refers to an alkyl group substituted with anaryloxy group.

The term “aralkyloxyalkyl-” refers to the group aryl-alk-O-alk- wherein“alk” is an alkylene group. “Lower aralkyloxyalkyl-” refers to suchgroups where the alkylene groups are lower alkylene.

The term “alkoxy-” or “alkyloxy-” refers to the group alkyl-O—.

The term “alkoxyalkyl-” or “alkyloxyalkyl-” refer to the groupalkyl-O-alk- wherein “alk” is an alkylene group. In “loweralkoxyalkyl-”, each alkyl and alkylene is lower alkylene.

The terms “alkylthio-” and “alkylthio-” refer to the groups alkyl-S—.

The term “alkylthioalkyl-” refers to the group alkyl-5-alk- wherein“alk” is an alkylene group. In “lower alkylthioalkyl-” each alkyl andalkylene is lower alkylene.

The term “alkoxycarbonyloxy-” refers to alkyl-O—C(O)—O—.

The term “aryloxycarbonyloxy-” refers to aryl-O—C(O)—O—.

The term “alkylthiocarbonyloxy-” refers to alkyl-S—C(O)—O—.

The terms “amido” or “carboxamido” refer to NR₂—C(O)— and RC(O)—NR¹—,where R and R¹ include H, alkyl, aryl, aralkyl, and alicyclic. The termdoes not include urea, —NR—C(O)—NR—.

The term “carboxamidoalkylaryl” and “carboxamidoaryl” refers to anaryl-alk-NR¹—C(O), and ar-NR¹—C(O)-alk-, respectively where “ar” isaryl, “alk” is alkylene, R¹ and R include H, alkyl, aryl, aralkyl, andalicyclic.

The term “hydroxyalkyl” refers to an alkyl group substituted with one—OH.

The term “haloalkyl” and “alkylhalide” refers to an alkyl groupsubstituted with one halo. Preferably, the halo is in the 2-position.

The term “cyano” refers to —C≡N.

The term “nitro” refers to —NO₂.

The term “acylalkyl” refers to an alkyl-C(O)-alk-, where “alk” isalkylene.

The term “aminocarboxamidoalkyl-” refers to the group NR₂—C(O)—N(R)-alk-wherein R is an alkyl group or H and “alk” is an alkylene group. “Loweraminocarboxamidoalkyl-” refers to such groups wherein “alk” is loweralkylene.

The term “heteroarylalkyl” refers to an alkyl group substituted with aheteroaryl group.

The term “perhalo” refers to groups wherein every C—H bond has beenreplaced with a C-halo bond on an aliphatic or aryl group. Suitableperhaloalkyl groups include —CF₃ and —CFCl₂.

The term “guanidino” refers to both —NR—C(NR)—NR₂ as well as —N═C(NR₂)₂here each R group is independently selected from the group of —H, alkyl,alkenyl, alkynyl, aryl, and alicyclic, all except —H are optionallysubstituted.

The term “amidino” refers to —C(NR)—NR₂ where each R group isindependently selected from the group of —H, alkyl, alkenyl, alkynyl,aryl, and alicyclic, all except —H are optionally substituted.

The term “pharmaceutically acceptable salt” includes salts of compoundsof formula I and its prodrugs derived from the combination of a compoundof this invention and an organic or inorganic acid or base. Suitableacids include HCl.

The term “prodrug” as used herein refers to any compound that whenadministered to a biological system generates a biologically activecompound as a result of spontaneous chemical reaction(s), enzymecatalyzed chemical reaction(s), and/or metabolic chemical reaction(s),or a combination of each. Standard prodrugs are formed using groupsattached to functionality, e.g. HO—, HS—, HOOC—, R₂N—, associated withthe drug, that cleave in vivo. Standard prodrugs include but are notlimited to carboxylate esters where the group is alkyl, aryl, aralkyl,acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters of hydroxyl,thiol and amines where the group attached is an acyl group, analkoxycarbonyl, aminocarbonyl, phosphate or sulfate. The groupsillustrated are exemplary, not exhaustive, and one skilled in the artcould prepare other known varieties of prodrugs. Such prodrugs of thecompounds of formula I, fall within the scope of the present invention.Prodrugs must undergo some form of a chemical transformation to producethe compound that is biologically active or is a precursor of thebiologically active compound. In some cases, the prodrug is biologicallyactive, usually less than the drug itself, and serves to improve drugefficacy or safety through improved oral bioavailability,pharmacodynamic half-life, etc. The biologically active compoundsinclude, for example, anticancer agents, antiviral agents, andantibiotic agents.

The term “bidentate” refers to an alkyl group that is attached by itsterminal ends to the same atom to form a cyclic group. For example,propylene imine contains a bidentate propylene group.

The structure

has a plane of symmetry running through the phosphorus-oxygen doublebond when R⁶═R⁶, V═W, W′═H, and V and W are either both pointing up orboth pointing down.

The term “cyclic 1′,3′-propane ester”, “cyclic 1,3-propane ester”,“cyclic 1′,3′-propanyl ester”, and “cyclic 1,3-propanyl ester” refers tothe following:

The phrase “together V and Z are connected via an additional 3-5 atomsto form a cyclic group containing 5-7 ring atoms, optionally containing1 heteroatom, substituted with hydroxy, acyloxy, alkoxycarbonyloxy, oraryloxycarbonyloxy attached to a carbon atom that is three atoms fromboth Y groups adjacent to V” includes the following:

The structure shown above (left) has an additional 3 carbon atoms thatforms a five member cyclic group. Such cyclic groups must possess thelisted substitution to be oxidized.

The phrase “together V and Z are connected via an additional 3-5 atomsto form a cyclic group, optionally containing one heteroatom, saidcyclic group is fused to an aryl group attached at the beta and gammaposition to the Y adjacent to V includes the following:

The phrase “together V and W are connected via an additional 3 carbonatoms to form an optionally substituted cyclic group containing 6 carbonatoms and substituted with one substituent selected from the groupconsisting of hydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy,and aryloxycarbonyloxy, attached to one of said additional carbon atomsthat is three atoms from a Y adjacent to V includes the following:

The structure above has an acyloxy substituent that is three carbonatoms from a Y, and an optional substituent, —CH₃, on the new 6-memberedring. There has to be at least one hydrogen at each of the followingpositions: the carbon attached to Z; both carbons alpha to the carbonlabeled “3”; and the carbon attached to “OC(O)CH₃” above.

The phrase “together W and W′ are connected via an additional 2-5 atomsto form a cyclic group, optionally containing 0-2 heteroatoms, and Vmust be aryl, substituted aryl, heteroaryl, or substituted heteroaryl”includes the following:

The structure above has V=aryl, and a spiro-fused cyclopropyl group forW and W′.

The term “phosphoramidite” refers to compounds attached via C, O, S, orN to the phosphorus in —P(YR)(YR) including cyclic forms, where Y isindependently —O— or —NR⁶—, but where at least one Y is —NR⁶—.

The term “phosphoramidate” refers to compounds attached via C, O, S, orN to the phosphorus in —P(O)(YR)(YR), including cyclic forms, where Y isindependently —O— or —NR⁶—, but where at least one Y is —NR⁶—.

The term “cyclic phosphoramidate” refers to phosphoramidates where—P(O)(YR)(YR) is

The carbon attached to V must have a C—H bond. The carbon attached to Zmust also have a C—H bond.

The term “carbocyclic sugar” refers to sugar analogs that contain acarbon in place of the oxygen normally found in the sugar ring. Itincludes 5-membered rings such as ribofuranosyl and arabinofuranosylsugars wherein the ring oxygen is replaced by carbon.

The term “acyclic sugar” refers to sugars that lack a ring, e.g.ribofuranosyl ring. An example is 2-hydroxyethoxymethyl in place of theribofuranosyl ring.

The term “L-nucleoside” refers to enantiomer of the naturalβ-D-nucleoside analogs.

The term “arabinofuranosyl nucleoside” refers to nucleoside analogscontaining an arabinofuranosyl sugar, i.e. where the 2′-hydroxyl ofribofuranosyl sugars is on the opposite face of the sugar ring.

The term “dioxolane sugar” refers to sugars that contain an oxygen atomin place of the 3′ carbon of the ribofuranosyl sugar.

The term “fluorinated sugars” refers to sugars that have 1-3carbon-fluorine atoms.

The term “P450 enzyme” refers to cytochrome enzymes that oxidize organiccompounds and includes naturally occurring P450 isozymes, mutants,truncated enzymes, post-transcriptase modified enzymes, and othervariants.

The term “liver” refers to liver and to like tissues and cells thatcontain the CYP3A4 isozyme or any other P450 isozyme found to oxidizethe phosphoramidates of the invention. Based on Example F, we have foundthat prodrugs of formula VI and VIII are selectively oxidized by thecytochrome P450 isoenzyme CYP3A4. According to DeWaziers et al. (J.Pharm. Exp. Ther., 253, 387-394 (1990)), CYP3A4 is located in humans inthe following tissues (determined by immunoblotting and enzymemeasurements):

Tissues % of liver activity Liver 100 Duodenum 50 jejunum 30 ileum 10colon <5 (only P450 isoenzyme found) stomach <5 esophagus <5 kidney notdetectableThus, “liver” more preferably refers to the liver, duodenum, jejunum,ileum, colon, stomach, and esophagus. Most preferably, liver refers tothe liver organ.

The term “hepatic” refers to the liver organ cells and not cells fromthe duodenum, for example.

The term “enhancing” refers to increasing or improving a specificproperty. It includes the situation where originally there was noactivity.

The term “liver specificity” refers to the ratio:

$\frac{\lbrack {{parent}\mspace{14mu}{drug}\mspace{14mu}{or}\mspace{14mu} a\mspace{14mu}{drug}\mspace{14mu}{metabolite}\mspace{14mu}{in}\mspace{14mu}{liver}\mspace{14mu}{tissue}} \rbrack}{\begin{bmatrix}{{{parent}\mspace{14mu}{drug}\mspace{14mu}{or}\mspace{14mu} a\mspace{14mu}{drug}\mspace{14mu}{metabolite}\mspace{14mu}{in}\mspace{14mu}{blood}},} \\{{urine},\mspace{14mu}{{or}\mspace{14mu}{other}\mspace{14mu}{non}\text{-}{hepatic}\mspace{14mu}{tissue}}}\end{bmatrix}}$as measured in animals treated with the drug or a prodrug. The ratio canbe determined by measuring tissue levels of the parent drug or drugmetabolite(s) including the biologically active drug metabolite or bothat a specific time or may represent an AUC based on values measured atthree or more time points.

The term “increased or enhanced liver specificity” refers to an increasein the liver specificity ratio in animals treated with the prodrugrelative to animals treated with the parent drug.

The term “enhanced oral bioavailability” refers to an increase of atleast 50% of the absorption of the dose of the parent drug or prodrug(not of this invention) from the gastrointestinal tract. More preferablyit is at least 100%. Measurement of oral bioavailability usually refersto measurements of the prodrug, drug, or drug metabolite in blood,tissues, or urine following oral administration compared to measurementsfollowing systemic administration.

The term “parent drug” refers to MH for phosphoramidates where M isconnected to —P(O)(YR)(YR) via oxygen, sulfur or nitrogen and to M-PO₃²⁻ when M is connected to —P(O)(YR)(YR) via carbon. For example, AZT canbe thought of as a parent drug in the form of MH. In the body AZT isfirst phosphorylated to AZT-PO₃ ²⁻ and then further phosphorylated toform AZT-triphosphate, which is the biologically active form. The parentdrug form MH only applies when M is attached via N, S, or O. In the caseof PMEA, the parent drug form is M-PO₃ ²⁻.

The term “drug metabolite” refers to any compound produced in vivo or invitro from the parent drug, or its prodrugs.

The term “pharmacodynamic half-life” refers to the time afteradministration of the drug or prodrug to observe a diminution of onehalf of the measured pharmacological response. Pharmacodynamic half-lifeis enhanced when the half-life is increased by preferably at least 50%.

The term “pharmacokinetic half-life” refers to the time afteradministration of the drug or prodrug to observe a diminution of onehalf of the drug concentration in plasma or tissues.

The term “therapeutic index” refers to the ratio of the dose of a drugor prodrug that produces a therapeutically beneficial response relativeto the dose that produces an undesired response such as death, anelevation of markers that are indicative of toxicity, and/orpharmacological side effects.

The term “enhancing the level of the biologically active drug” refers toincreasing the level of the biologically active drug in the liverrelative to the level achieved after administration of the parent drugwhere the same mode of administration is used for both the prodrug andthe parent drug.

The term “cancer expressing a P450 enzyme” refers to tumor cells havinga specific activity, either with or without an agent that induces theP450 activity in the tumor cells, of at least 5% of the liver specificactivity.

The term “low levels of enzymes” refers to levels below that necessaryto produce the maximal response of the biologically active agent.

The term “inadequate cellular production” refers to levels that arebelow that necessary to produce the maximal response of the biologicallyactive agent. For example, the response may be viral titre.

The term “sustained delivery” refers to an increase in the period inwhich there is a prolongation of therapeutically-effective drug levelsdue to the presence of the prodrug.

The term “bypassing drug resistance” refers to the loss or partial lossof therapeutic effectiveness of a drug (drug resistance) due to changesin the biochemical pathways and cellular activities important forproducing and maintaining the biologically active form of the drug atthe desired site in the body and to the ability of an agent to bypassthis resistance through the use of alternative pathways and cellularactivities.

The term “FBPase inhibitors” refers to compounds that inhibit the humanenzyme fructose 1,6-bisphosphatase with an IC50 of at least 100 μM andlower glucose in a normal 18-hour fasted rat following a 100 mg/kg dosei.v. The biologically active FBPase inhibitors are M-PO₃ ²⁻ wherein M isconnected via a carbon, or via an oxygen when MH is a imidazolecontaining nucleoside analog.

The term “biologically active drug or agent” refers to the chemicalentity that produces the biological effect. In this invention,biologically active agents refers to M-PO₃ ²⁻, M-P(O—)NHR⁶⁻, MP₂O₆ ³⁻,or MP₃O₉ ⁴⁻ where M can be the same M as in the parent drug or ametabolite.

The term “therapeutically effective amount” refers to an amount that hasany beneficial effect in treating a disease or condition.

The term “decreased activity of the phosphorylating enzyme” includesdecreased production of the enzyme, mutations that lead to decreasedefficiency (lower K_(m) or V_(max)), or enhanced production ofendogenous inhibitors of the enzyme.

The term “phosph(on)ate” refers to compounds attached via C, O, S, or Nto PO₃ ²⁻ or P(O)(NHR⁶)O⁻. The counterion may be H⁺ or a metal cation.

The term “nucleoside” refers to a purine or pyrimidine base, includinganalogs thereof, connected to a sugar, including heterocyclic andcarbocyclic analogs thereof.

The following well known drugs are referred to in the specification andthe claims. Abbreviations and common names are also provided.

araA; 9-b-D-arabinofuranosyladenine (Vidarabine)

AZT; 3′-azido-2′,3′-dideoxythymdine (Zidovudine)

d4T; 2′,3′-didehydro-3′-deoxythymidine (Stavudine)

ddI; 2′,3′-dideoxyinosine (Didanosine)

ddA; 2′,3′-dideoxyadenosine

ddC; 2′,3′-dideoxycytidine (Zalcitabine)

L-ddC; L-2′,3′-dideoxycytidine

L-FddC; L-2′,3′-dideoxy-5-fluorocytidine

L-dT; b-L-thymidine (NV-02B)

L-dC; b-L-2-deoxycytidine (NV-02C)

L-d4C; L-3′-deoxy-2′,3′-didehydrocytidine

L-Fd4C; L-3′-deoxy-2′,3′-didehydro-5-fluorocytidine

3TC; (−)-2′,3′-dideoxy-3′-thiacytidine;2′R,5′S(−)-1-[2-(hydroxymethyl)oxathiolan-5-yl]cytosine (Lamivudine)

1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Ribavirin)

FIAU; 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-5-iodouridine

FIAC; 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-5-iodocytosine

BHCG; (±)-(1a,2b,3a)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine

FMAU; 2′-Fluoro-5-methyl-b-L-arabino-furanosyluracil

BvaraU; 1-b-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (Sorivudine)

E-5-(2-bromovinyl)-2′-deoxyuridine

TFT; Trifluorothymidine (Trifluorothymidine)

5-propynyl-1-arabinosyluracil (Zonavir)

CDG; carbocyclic 2′-deoxyguanosine

DAPD; (−)-B-D-2,6-diaminopurine dioxolane

FDOC; (−)-B-D-5-fluoro-1-[2-(hydroxymethyl)-1,3-dioxolane]cytosine

d4C; 3′-deoxy-2′,3′-didehydrocytidine

DXG; dioxolane guanosine

FEAU; 2′-deoxy-2′-fluoro-1-b-D-arabinofuranosyl-5-ethyluracil

FLG; 2′,3′-dideoxy-3′-fluoroguanosine

FLT; 3′-deoxy-3′-fluorothymidine

FTC; (−)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine

5-yl-carbocyclic 2′-deoxyguanosine (BMS200,475)

[1-(4′-hydroxy-1′,2′-butadienyl)cytosine] (Cytallene)

Oxetanocin A;9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanosyl)adenine

Oxetanocin G;9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanosyl)guanine

ddAPR: 2,6-diaminopurine-2′,3′-dideoxyriboside

3TC; (−)-2′,3′-dideoxy-3′ thiacytidine; (2R,5S)1-[2-(hydroxymethyl)-1,3-oxathiolane-5-yl]cytosine (Lamivudine)

Cyclobut A; (+/−)-9-[(1 beta,2 alpha,3beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine

Cyclobut G; (+/−)-9-[(1 beta,2 alpha,3beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine (Lobucavir)

5-fluoro-2′-deoxyuridine (Floxuridine)

dFdC; 2′,2′-difluorodeoxycytidine (Gemcitabine)

araC; arabinosylcytosine (Cytarabine)

bromodeoxyuridine

IDU; 5-iodo-2′-deoxyuridine (Idoxuridine)

CdA; 2-chlorodeoxyadenosine (Cytadribine)

F-ara-A; fluoroarabinosyladenosine (Fludarabine)

ACV; 9-(2-hydroxyethoxylmethyl)guanine (Acyclovir)

GCV; 9-(1,3-dihydroxy-2-propoxymethyl)guanine (gancyclovir)

9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (Penciclovir)

(R)-9-(3,4-dihydroxybutyl)guanine (Buciclovir)

phosphonoformic acid (Foscarnet)

PPA; phosphonoacetic acid

PMEA; 9-(2-phosphonylmethoxyethyl) adenine (Adefovir)

PMEDAP; 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine

HPMPC; (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (Cidofovir)

HPMPA; (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine

FPMPA; 9-(3-fluoro-2-phosphonylmethoxypropyl) adenine

PMPA; 9-[2-(R)-phosphonylmethoxy)propyl]adenine (Tenofovir)

araT; 9-b-D-arabinofuranosylthymidine

FMdC; (E)-2′-deoxy-2′(fluoromethylene)cytidine

AICAR; 5-aminoimidazole-4-carboxamido-1-ribofuranosyl

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to the use of new cyclic phosphoramidatemethodology which allows compounds to be efficiently converted tophosph(on)ate containing compounds by P450 enzymes found in largeamounts in the liver and other tissues containing these specificenzymes. This methodology can be applied to various drugs and todiagnostic imaging agents. More specifically, the invention is directedto the use of prodrugs that undergo non-esterase-mediated hydrolysisreactions to produce MP(O)(NHR⁶)O⁻ which is either biologically activeor is transformed into the biologically active agent M-PO₃ ²⁻, M-P₂O₆ ³⁻or M-P₃O₉ ⁴⁻. Because highly charged phosph(on)ate containing compoundsare not readily absorbed in the gastrointestinal tract, this prodrugmethodology can be used to enhance absorption after oral administration.

In another aspect of the invention, this prodrug methodology can also beused to enhance the pharmacodynamic half-life relative to the parentdrug because the cyclic phosph(on)ates of the invention can avoidmetabolism by enzymes which metabolize the parent drug. Similarly,prodrugs of the invention can enhance the pharmacodynamic half-life ofthe phosph(on)ate-containing compound because the prodrug avoidsclearance pathways used to clear negatively-charged compounds from thebloodstream.

In another aspect of the invention, this prodrug methodology can also beused to achieve sustained delivery of the parent drug because oxidationof the novel prodrugs proceeds in the liver at different rates andtherefore selection of prodrugs that oxidize slowly but at a ratesuitable for maintaining therapeutically effective levels will produce asustained therapeutic effect.

The novel cyclic 1,3-propanylester methodology of the present inventionmay also be used to increase the distribution of a particular drug orimaging agent to the liver which contains abundant amounts of the P450isozymes responsible for oxidizing the cyclic 1,3-propanylester of thepresent invention so that the free phosph(on)ate is ultimately produced.Accordingly, this prodrug technology should prove useful in thetreatment of liver diseases or diseases where the liver is responsiblefor the overproduction of the biochemical end product such a glucose,cholesterol, fatty acids and triglycerides. Such diseases include viraland parasitic infections, liver cancer, liver fibrosis, diabetes,hyperlipidemia, and obesity. In one aspect, preferably the disease isnot diabetes. In addition, the liver specificity of the prodrugs shouldalso prove useful in the delivery of diagnostic agents to the liver.High levels of the biologically active drug are achieved in tissuesexpressing P450 enzymes capable of cleaving the prodrug due tosignificant retention by these cells of the activated prodrug,subsequent prodrug cleavage intermediates as well as the biologicallyactive drug. In addition, high levels of the biologically active drugare achieved relative to the levels achieved after administration of theparent drug when the parent drug is poorly phosphorylated in the tissueor has a relatively short pharmacokinetic half-life due to metabolism orother clearance mechanisms.

The use of certain prodrug analogs of cyclophosphamide are alsoenvisioned for treatment of cancers of the GI tract (e.g. primary andsecondary liver cancers) as well as cancers where cytochrome P450s areintroduced artificially, e.g. by retrovirus and other well known genetherapy strategies as well as via implanted cells engineered to expresscytochrome P450s.

These specific P450 enzymes are also found in other specific tissues andcells, and thus this methodology may also be used to increase thedelivery of these agents to those tissues.

In another aspect of the invention, the characteristic that most of thecyclic phosphoramidates of the present invention are metabolized in theliver to produce the phosph(on)ate can enable the use of the prodrugmethodology of the present invention to increase the therapeutic indexof various drugs which have side effects related to the amount of thedrug or its metabolites which are distributed in extrahepatic tissues.Increased therapeutic index can also result from increased liver levelsof the biologically active agent and therefore greater efficacy relativeto the parent drug administered at similar doses.

In another aspect of the invention, prodrugs can be used to deliverphosph(on)ates to tissues that generate suboptimal levels ofphosph(on)ates and their associated higher phosphorylated metabolitesdue to poor substrate activity of the parent compound and/or poorexpression of enzymes used to phosphorylate the parent compound. Poorexpression of the phosphorylating enzyme occurs naturally or from downregulation of the phosphorylating enzyme following chronicadministration of the drug or other agents.

In another aspect of the invention, novel phosphoramidite andphosphoramidate intermediates are described.

In another aspect, methods of preparing the cyclic phosphoramidateprodrugs are described.

These aspects are described in greater detail below.

Phosphoramidate Drugs:

Compounds of the formula M-P(O)(NHR⁶)O⁻ are useful compounds for bindingto nucleotide binding sites, such as AMP-binding sites, and certainsites known to recognize negatively charged compounds, e.g. carboxylicand phosphonic acids. Enzymes that catalyze the addition of water to acarbonyl compound, specifically a peptide carbonyl, an ester, a ketoneor an aldehyde, are of particular interest since these enzymes recognizecompounds that have a tetrahedral group containing a negatively chargedoxygen. An example is the zinc metalloproteinase class of enzymes whichadd water across a peptide carbonyl using a zinc-assisted catalyticmechanism. These enzymes are inhibited by phosphoramidates, e.g. NEP24.11 is inhibited by the natural product Phosphoramidon. The prodrugstrategy can provide a useful means for delivery of these compoundsorally, or delivery of certain compounds to the liver in order toachieve greater efficacy or a greater therapeutic window. Enzymesinhibitors that may be suitable for delivery as prodrugs include certainphosphoramidates that inhibit NEP24.11, collagenase, stromolysin,gelatinase, ACE, endothelin converting enzyme, and metalloproteinasesinvolved in matrix remodeling such as occurs in Rheumatoid arthritis andosteoarthritis and in the heart following an acute myocardialinfarction, and tumor metastasis.

Phosphonic Acids and Phosphoric Acids:

Cleavage of the prodrug of formula I produces the phosphoramidateM-P(O)(NHR)O⁻ which in some cases is further converted to thecorresponding M-PO₃ ²⁻ via either chemical hydrolysis or enzymecatalyzed hydrolysis (phosphatases, amidases). The product M-PO₃ ²⁻ caneither be the drug or itself may be further metabolized to producehigher order phosphates, e.g. M-P₂O₆ ³⁻ or M-P₃O₉ ⁴⁻ via cellularkinases. In some cases the intermediate M-P(O)(NHR)O⁻ may be convertedto higher order phosphates via initial conversion to M-H.

Enhancing Oral Bioavailability

The invention pertains to certain cyclic phosphoramidates and their useto deliver, most preferably via oral administration, a therapeuticallyeffective amount of the corresponding phosph(on)ate compounds,preferably to an animal in need thereof. Prodrugs of the inventionenhance oral bioavailability of certain drugs by changing the physicalproperty of the drug as a consequence of the prodrug moiety and itssubstituents V, Z, W, and W′. The active drug may be M-P(O)(NHR⁶)O⁻ orM-PO₃ ²⁻. Alternatively, both may instead undergo further metabolism,e.g. phosphorylation by kinases to form M-P₂O₆ ³⁻ and/or M-P₃O₉ ⁴⁻ asthe active drug substance.

Compounds containing a free phosphonic acid or a phosphoric acid groupgenerally exhibit poor (<2%) oral bioavailability since these groups arehighly charged at physiological pH. Charged groups on compounds withmolecular weights greater than 250 Daltons impede passive diffusionacross cell membranes as well as absorption across the gut epithelialcell layer. Neutral prodrugs of these compounds have therefore beenstudied since these compounds would be more lipophilic and thereforemore likely to exhibit improved intestinal permeability. Although manyprodrug classes have been reported, few have been found that exhibitproperties suitable for drug development.

The most common prodrug class, and the class almost exclusively used forclinical candidates, is the acyloxyalkyl esters. These prodrugs,however, often exhibit only a modest improvement in oral bioavailabilitydue to poor aqueous stability, poor stability to acidic/basic pH andrapid degradation by esterases in the gastrointestinal tract (Shaw &Cundy, Pharm. Res. 10, (Suppl), S294 (1993). Another class of prodrugsare the bis-aryl prodrugs (e.g. DeLombert et al. J. Med. Chem. 37, 498(1994)) which have shown in a few isolated cases to provide good tomodest improvements in oral bioavailability. The major limitation withthis class of compounds is that the prodrug ester often is degraded tothe monoacid rapidly in vivo but conversion to the parent drug occursonly slowly (sometimes over days) if at all.

The prodrugs of the invention exhibit improved properties that lead toenhanced oral bioavailability relative to the parent drug. Severalcharacteristics of the present cyclic phosphoramidate prodrugs maycontribute to their ability to enhance oral bioavailability. First, theprodrugs exhibit good stability in aqueous solutions across a wide rangeof pHs. (Example A) This pH stability prevents immediate hydrolysis inthe mouth and GI tract prior to absorption. The pH stability can also bebeneficial during formulation of the product.

Second, the prodrugs are resistant to esterases and phosphatases whichare abundant in the gastrointestinal tract. Because much of theadministered dose remains intact in the G.I. tract, the compound remainsless highly charged than a free phosph(on)ate which means more of thedrug can be absorbed by passive diffusion and enter the blood stream.(Example B)

Last, the prodrug can limit metabolism at other sites on the molecule.For example, the prodrugs of the invention eliminate metabolism of thepurine base of araA by adenosine deaminase which is also abundant in theGI tract. (Example B) The amine of araA which is normally deaminated bythe enzyme is protected by the cyclic phosphate moiety which is locatedon the 5′-ribofuranosyl hydroxyl. Reduced metabolism at other sites ofthe molecule enables more of the drug to circulate in the blood stream.Although not all of these properties will be applicable to every prodrugof every drug, each of these properties can enable more drug to survivethe GI tract and be available for absorption.

The novel prodrug strategy of the invention will be useful for the oraldelivery of drugs that act in the liver as well as certain drugs thatact on targets located in the vascular system or extrahepatic tissues.Because the highest concentration of CYP3A4 (the enzyme responsible foractivating the novel prodrugs) is in the liver, the biologically activedrug has a high concentration in the liver, relative to other tissues.In one aspect, parent drugs which act in the liver are preferred.

However, some of the phosph(on)ates are exported by organic aniontransporters in the liver and enter the blood stream. Manyphosph(on)ates in the blood stream are cleared quickly by the kidneys.Such compounds probably will not reach therapeutic levels inextrahepatic tissues. However, there are some phosph(on)ates andphosphates that are able to remain in circulation because they are notrapidly cleared by the kidneys (e.g. NEP inhibitors). Such compounds areable to achieve therapeutically effective levels in blood andextrahepatic tissues. Thus, in another aspect, oral delivery toextrahepatic tissues of phosph(on)ates which are not cleared by thekidneys is preferred. Thus, such parent drugs that act at sitesaccessible to the free phosph(on)ic acid such as targets within thevasculature system, or enzyme or receptor targets that are located oncell membranes which are exposed to the blood or fluid in theintrastitial space are preferred. Targets suitable for this aspect ofthe invention would be targets in which the phosphonic acid administeredparenterally (e.g. via i.v. injection) produces a pharmacological orbiochemical response expected to be useful for treating a diseasecondition.

Since the inhibitors exhibit poor oral bioavailability (<2%), prodrugsof the type described in this invention could enhance the oralbioavailability and produce the phosphonic acid following prodrugcleavage in the liver. Suitable circulating drug levels are expectedafter prodrug cleavage in the liver, since the liver is known to excretephosphonic acids into the circulation.

Oral bioavailability can also be calculated by comparing the area underthe curve of prodrug, parent drug, and/or metabolite concentration overtime in plasma, liver, or other tissue or fluid of interest followingoral and i.v. administration. (Example P)

For example, for drugs excreted renally in large amounts, oralbioavailability can be measured by comparing the amount of the parentdrug or metabolite excreted in the urine, for example, after oral andi.v. administration of the prodrug. A lower limit of oralbioavailability can be estimated by comparison with the amount of parentdrug excreted in the urine after administration of the prodrug (p.o.)and the parent drug (i.v.). Prodrugs of the invention show improved oralbioavailability across a wide spectrum of prodrugs, with many preferablyshowing increases of 1.5 to 10-fold in oral bioavailability. Morepreferably, oral bioavailability is enhanced by at least 2-fold comparedto the parent drug.

Agents are known that inhibit CYP3A4 in the gastrointestinal tract. Forexample, grapefruit juice is known to decrease the activity putativelyvia a component in the grapefruit juice (e.g. Bergamottin; Chem ResToxicol 1998, 11, 252-259) which results in the inactivation and/or downregulation of the enzyme. Since only GI CYP3A4 is affected, the oralabsorption of the prodrugs of this invention should be enhanced. Acombination of agents that inhibit, inactivate, or downregulate P450sthat metabolize the prodrugs will have the effect of enhancing theirabsorption and thereby making more prodrug available for metabolism inthe liver. The net effect of the combination would therefore be todeliver more drug to the liver after oral absorption.

Sustained Delivery

Drugs that undergo rapid elimination in vivo often require multipleadministrations of the drug to achieve therapeutically-effective bloodlevels over a significant period of time. Other methods are alsoavailable including sustained release formulations and devices. Prodrugsthat breakdown over time can also provide a method for achievingsustained drug levels. In general, this property has not been possiblewith the known phosph(on)ate prodrugs since either they undergo rapidhydrolysis in vivo (e.g. acyloxyalkyl esters) or very slow conversion(e.g. di-aryl prodrugs).

The cyclic phosphoramidates of the invention are capable of providingsustained drug release by providing a steady release of the drug overtime. For example, most phosphates undergo dephosphorylation in vivowithin minutes after systemic administration via the action ofphosphatases present in the blood. Similarly, acyloxyalkyl esters ofthese phosphates undergo rapid esterase-mediated hydrolysis to thephosphate which then is rapidly dephosphorylated. Some prodrugs of thecurrent invention may enable prolonged drug delivery since many of thepresent prodrugs are oxidized slowly over time to the phosph(on)ate inthe livers (Example S). Suitably positioned, prodrug moieties canprevent or slow systemic metabolism associated with the parent drug.

Sustained delivery of the drugs is achievable by selecting the prodrugsof formula I that are hydrolyzed in vivo at a rate capable ofmaintaining therapeutically effective drug levels over a period of time.The cleavage rate of the drug may depend on a variety of factors,including the rate of the P450 oxidation, which is dependent on both thesubstituents on the prodrug moiety, the stereochemistry of thesesubstituents and the parent drug. Moreover, sustained drug productionwill depend on the rate of elimination of the intermediate generatedafter oxidation and the rate and availability of the prodrug to theliver, which is the major site of oxidation. Identification of theprodrug with the desired properties is readily achieved by screening theprodrugs in an assay that monitors the rate of drug production in thepresence of the major P450 enzyme involved in the metabolism, in thepresence of liver microsomes or in the presence of hepatocytes. Theseassays are illustrated in Examples C, D, F, G, I, J, respectively.

It is contemplated that prodrugs of the present invention could becombined to include, for example, one prodrug which produces the activeagent rapidly to achieve a therapeutic level quickly, and anotherprodrug which would release the active agent more slowly over time.

Improved Pharmacodynamic Half-Life

The pharmacodynamic half-life of a drug can be extended by the novelprodrug methodology as a result of both its ability to produce drug overa sustained period and in some cases the longer pharmacokinetichalf-life of the prodrug. Both properties can individually enabletherapeutic drug levels to be maintained over an extended periodresulting in an improvement in the pharmacodynamic half-life. Thepharmacodynamic half-life can be extended by impeding the metabolism orelimination pathways followed by the parent drug. For some drugs, theprodrugs of the present invention are able to avoid the metabolism orelimination pathways associated with the parent drug and thereby existas the prodrug for extended periods in an animal. High levels of theprodrug for an extended period result in sustained production of theparent drug which can result in an improvement in the drugpharmacodynamic half-life.

An example of the ability of the prodrug class to impede metabolicpathways associated with the parent drug is shown by the araAMPprodrugs. In comparison to araAMP, prodrugs show no ara-hypoxanthine(“araH”) which is the known metabolic byproduct of araA produced in e.g.plasma and the gastrointestinal tract after oral or i.v./administration.AraAMP on the other hand is rapidly and nearly completely converted toaraH, which is produced by first dephosphorylation to araA viaphosphatases followed by deamination of the base via adenosinedeaminase. The prodrug moiety prevents both dephosphorylation anddeamination from occurring.

A common route of elimination of phosph(on)ate drugs is via the kidneysand a transporter that recognizes anionic compounds. Completeelimination of phosphonate and phosphate containing drugs from thecirculation often occurs only minutes after drug administration. Theprodrugs of this invention slow the elimination of these drugs bymasking the negative charge until after oxidation and hydrolysis inliver and like tissues.

Enhanced Selective Delivery of Agents to the Liver and Like Tissues

Delivery of a drug to the liver with high selectivity is desirable inorder to treat liver diseases or diseases associated with the abnormalliver properties (e.g. diabetes, hyperlipidemia) with minimal sideeffects.

Analysis of the tissue distribution of CYP3A4 indicates that it islargely expressed in the liver (DeWaziers et al., J. Pharm. Exp. Ther.253: 387 (1990)). Moreover, analysis of tissue homogenates in thepresence of prodrugs indicates that only the liver homogenate cleavesthe prodrug and to a lesser degree homogenates from tissues in the upperG1. Kidney, brain, heart, stomach, spleen, muscle, lung, and testesshowed no appreciable cleavage of the prodrug (Example D).

Evidence of the liver specificity was also shown in vivo after both oraland i.v. administration of the prodrugs. Administration of a prodrug ofaraAMP i.v. gives liver levels of the bioactive drug araATP greater thanachieved by an equivalent dose of either araA or araAMP. In contrast,the prodrug fails to produce detectable amounts of the araA by-productaraH, which, as reported in the literature, is readily detected aftereither araA or araAMP administration. Similarly, the prodrug achieveshigh liver levels without production of the metabolite araH after oraladministration. Since the prodrugs are cleaved by liver abundantenzymes, oral administration may enable even higher liver specificityvia a first pass effect.

The prodrugs described in this invention can be tailored such that theelimination step is fast and therefore the product is produced near thesite of oxidation, which for these prodrugs is in the liver or otherP450-expressing tissue/cells.

In some cases liver specificity will be achieved most optimally usingprodrugs of highly reactive drugs, which after production, act locallyat a fast rate relative to diffusion out of the liver.

Agents that induce P450 activity, e.g. CYP3A4 activity, are known. Forexample, rifampicin, glucocorticoids, phenobarbital, erythromycin areknown to enhance CYP3A4 activity in rat and human livers following. P450activity can be monitored in humans by non-invasive methods e.g. via[14C] erythromycin breath test. These studies are useful in theidentification of agents that activate CYP3A4 in humans. Accordingly,for prodrugs where drug delivery is limited by prodrug metabolism rate(e.g. rate of clearance of prodrug is fast relative to rate of prodrugcleavage), agents such as rifampicin can be used in combination or asadjuncts or pre-treatments to enhance CYP3A activity in the liver andthereby to increase liver drug levels. (Examples J and O)

Prodrug Cleavage Mechanism

The prodrugs of the current invention are simple, low molecular weightmodifications of the drug which enable liver-selective drug delivery onthe basis of the their sensitivity to liver-abundant enzymes. Theprodrug cleavage mechanism is postulated to entail an oxidation andβ-elimination based on studies analyzing reaction requirements andproducts. In some cases, M-P(O)(NR⁶)O⁻ is further metabolized to M-PO₃²⁻ and M-P₃O₉ ⁴⁻. Prodrugs are stable to aqueous solution across a broadpH range and therefore do not undergo a chemical cleavage process toproduce the parent drug. In addition the prodrugs are stable toesterases and blood proteins. In contrast, the prodrugs are rapidlycleaved in the presence of liver microsomes from rats (Example C) andhumans (Examples D and F). The drug is also produced in freshly isolatedrat hepatocytes where it is detected as the corresponding phosph(on)ateand/or biologically active agent (Examples G and J). Moreover, when theparent drug is an FBPase inhibitor, the production of the drug issupported by the ability of the prodrug to result in potentgluconeogenesis inhibition (Example I).

Possible specific enzymes involved in the cleavage process wereevaluated through the use of known cytochrome P450 inhibitors (ExampleE). The studies indicate that the isoenzyme cytochrome CYP3A4 isresponsible based on ketoconozole inhibition of drug formation.

The biologically active agent is detected in the liver followingadministration of drugs of formulae VI-VIII, shown below:

Prodrugs of the following formulas are particularly preferred.

Prodrugs of formula VI cleave to generate aryl vinyl ketone whereasprodrugs of formula VIII cleave to generate phenol (Example L). Themechanism of cleavage could proceed by the following mechanisms, shownfor compounds where one Y is NR⁶.

Although the esters in the invention are not limited by the abovemechanisms, in general, each ester contains a group or atom susceptibleto microsomal oxidation (e.g. alcohol, benzylic methine proton), whichin turn generates an intermediate that breaks down to the parentcompound in aqueous solution via β-elimination of the M-P(O)(YH)₂. Thisspecies is either the active component or is further transformed viachemical or enzymatic processes to M-PO₃ ²⁻, M-P₂O₆ ³⁻, or M-P₃O₉ ⁴⁻.Furthermore, although these specific prodrugs are cleaved by CYP3A4,other prodrugs in the class may be substrates for other P450s. Smallchanges in structure are known to influence substrate activity and P450preference. The identification of the isoenzyme(s) activating theprodrug is accomplished according to the procedure described in ExampleB.

Alternatively, cyclic phosphoramidates can serve as a prodrug ofphosph(on)ates or higher order phosph(on)ates since intermediatephosphoramidates can be converted to MPO₃ ²⁻, MP₂O₆ ³⁻, or MP₃O₉ ⁴⁻ viathe action of phosphatases/amidases which can produce MH or MPO₃ ²⁻which in turn can be phosphorylated by kinases to the biologicallyactive.

compound.Increased Therapeutic Index

The prodrugs of this invention can significantly increase thetherapeutic index (“TI”) of certain drugs. In many cases, the increasedTI is a result of the high liver specificity. For example, araA andaraAMP are known to produce significant systemic side effects and theseside effects are associated with blood levels of the araA byproductaraH. Presumably the side effects are a result of toxicities of araH oraraA in extrahepatic tissues (e.g. nerves) which produce e.g. theneuropathies associated with the drug in man (>40% of patients receivingaraA). Prodrugs of araA show a substantial shift in the liver(araATP)/urine (araH) ratio in comparison with araAMP.

Renal toxicity is a common toxicity associated with phosphonic acids.The toxicity results from transport, e.g. via the organic aniontransporters located on the basolateral membrane of the renal proximaltubule, of the negatively charged drug into e.g. tubular cells whichthen accumulate the drug to high concentrations unless there is anequally efficient transport of the drug out of the cell via luminaltransport mechanisms (e.g., anion exchange or facilitated diffusion).Many examples have been reported in the literature of nephrotoxicphosphonic acids, e.g. PMEA and HPMPA. The novel prodrug of PMEA showsonly small amounts of PMEA in the urine relative to either PMEA orbisPOM PMEA at doses that achieved comparable liver drug levels.

Another common toxicity associated with phosphonic acid drugs isgastrointestinal toxicity via in some cases GI erosions. Prodrugs of thecurrent invention can decrease GI toxicities, especially toxicitiesproduced by direct action of the drug on the GI tract after oraladministration. Similar to the kidney, gut epithelial cells have organicanion transporters which can result in high intracellular drug levels ancytotoxicity. Since the negatively charged phosph(on)ate is not revealeduntil after absorption and cleavage in the liver, prodrugs of thisinvention reduce gut toxicity.

Severe toxicities are also associated with nearly all anticancer agents.In an effort to decrease these toxicities during treatment of primary orsecondary liver cancers, drugs are sometimes administered directly intothe portal artery in order to increase liver drug exposure. Sinceoncolytic drugs typically are associated with significant side effects,local administration enables greater hepatic uptake and therebydecreased extrahepatic toxicities. To further increase liver uptake,chemoembolization is sometimes used in conjunction with hepatic arterydrug infusion. The high liver specificity of the prodrugs in the currentinvention suggest that systemic side effects will be similarly minimizedby the novel prodrug approach.

Moreover, primary and secondary liver cancers are particularly resistantto both chemotherapy and radiotherapy. Although the mechanism for theresistance is not completely understood, it may arise from increasedliver gene products that lead to rapid metabolism and/or export ofchemotherapeutic agents. In addition, the liver, which is generallyassociated with xenobiotic metabolism and generation of cytotoxicintermediates, is equipped by nature with multiple protective mechanismso that damage from these intermediates are minimized. For example, theintracellular concentration of glutathione is very high in the liverrelative to other tissues presumably so that intermediates capable ofalkylating proteins and DNA are detoxified through a rapid intracellularreaction. Consequently, the liver may be resistant to chemotherapeuticagents because of these mechanisms and therefore require higher thannormal concentrations of the oncolytic agent to achieve success. Higherliver concentrations require higher doses of the drug which commonlyresult in extrahepatic toxicities.

Prodrugs of the current invention can be effective against these cancersbecause they enable higher levels of the biologically active drugthrough a variety of mechanisms. For example, the high liver specificitycan enable higher doses since the dose-limiting extrahepatic sideeffects are reduced. Second, sustained delivery of the prodrug canenable high drug levels to be maintained over a sustained period. Third,in some cases, the parent drug is poorly phosphorylated in the targetcells. The prodrugs of this invention can bypass this step and deliverthe phosph(on)ate which in some cases is further metabolized to thebiologically active agent. Last, the therapeutic index can sometimes befurther enhanced by co-administration of CYP inducers (e.g. rifampicin,glucocorticoids, phenobarbital, erythromycin) which can enhanceconversion in the liver and thereby enable higher levels of thebiologically active drug without increasing the dose (Examples J and O).These benefits of the prodrugs enable successful therapy of liverdiseases, such as primary and secondary liver cancers as well as otherliver diseases.

Byproduct Toxicity

Prodrugs of the invention are also useful because they generatebyproducts that are either non-toxic or rapidly detoxified. Some of thebyproducts generated by prodrugs of the invention are not considered tobe highly toxic at doses expected to be used in the clinic, especiallygiven that the byproduct would be generated in the liver (e.g. phenol).Other byproducts generated by the prodrugs of this invention, e.g. arylvinyl ketone, can be considered as alkylating agents and therefore couldproduce either or both cytotoxicity and genotoxicity. These toxicities,however, are eliminated for prodrugs activated in the liver since: 1)the byproduct is produced at or near the site of prodrug activation; and2) natural defense mechanisms exist at the site of byproduct generationand are able to completely detoxify the byproduct. Other mechanisms mayalso assist in the detoxification of the byproduct, including reactionsthat lead to oxidation, reduction or transformation (e.g. sulfation) ofthe byproduct.

The primary defense system expected to protect cells from the prodrugbyproduct involves the natural thiol glutathione and possibly an enzymethat catalyzes the addition of glutathione to electrophiles, i.e.glutathione S-transferase (GST). Glutathione and GST are alwaysco-expressed with CYPs as part of a natural defense mechanism thatprotects the cell from highly reactive oxygen species generated duringCYP-mediated oxidation of organic compounds. CYPs are expressed incertain cells (primarily hepatocytes) to oxidize organic compounds andthereby assist in their elimination from the body. Tissue distributionstudies show that CYP3A4 as well as most other P450s are expressedhighest in the liver followed by the small intestine. All other tissueshave <<2% of the liver CYP3A4 activity. Similarly, analysis ofglutathione levels indicates that the liver (10 mM) and GI have nearly1000-fold higher glutathione levels than other tissues. These levels ofglutathione are still high even in diseased livers.

Glutathione reacts rapidly with vinyl ketones, and any alkylating agent,to generate a conjugate which is then eliminated via the bile or kidney.Alkylation of other proteins or nucleic acids in the cell is notexpected in cells with glutathione since glutathione is a much betternucleophile than heteroatoms on the bases of nucleic acids and exists atmuch higher concentration than thiol groups on the surface of proteins.

By products such as acrolein can produce bladder toxicity which theseprodrugs can prevent by their ability to generate the byproduct withinthe hepatocyte. Examples M and N are useful for testing for byproducttoxicity, which is not expected in the liver as long as hepaticglutahione levels remain >20% of normal. Glutathione is readily measuredin hepatocytes as well as in vivo (Examples M and N). Cytotoxicity isdetected in hepatocytes via testing of cell viability with trypan blueand by analyzing for elevation of live enzymes. In vivo, liver toxicityis evaluated by analysis of liver enzyme elevation in the plasma.

Non-Mutagenic Prodrugs

Prodrugs of the invention are elevated by a postulated mechanisminvolving an initial oxidation followed by a β-elimination reaction. Insome cases, e.g. certain prodrugs of formula VI and formula VII, thebiproduct of the reaction is an α,β-unsaturated carbonyl compound, e.g.vinyl phenyl ketone for prodrugs where V=Ph, Z, W and W′=H. Compoundscan act as Michael acceptors and react with nucleophiles via a Michaeladdition. Mutagenesis is observed with some α-β-unsaturated ketones andcertain toxicities arise from Michael addition adducts (e.g. acroleinproduces bladder toxicities). The degree to which these activities limitthe use of compounds of Formula VI is dependent on the severity of thetoxicity and the indicated disease.

Prodrugs that produce non-toxic and non-mutagenic biproducts areespecially preferred for the treatment of chronic diseases (e.g.diabetes). Frequently, it is difficult to predict the mutagenicproperties of a compound. For example, a number of acrylates have beenshown to produce positive mutagenic responses as indicated by increasedchromosome aberrations and micronucleus frequencies in cultured L5179Ymouse lymphoma cells (Dearfield et al., Mutagenesis 4, 381-393 (1989)).Other acrylates, however, are negative in this test (J. Tox. Envir.Health, 34, 279-296 (1991)) as well as in the Ames test and the CHOassay which measures newly induced mutations at the hypoxanthine-guaninephosphoribosyltransferase (hgprt) locus (Mutagenesis 6, 77-85 (1991)).Phenyl vinyl ketone lacks teratogenic activity in rat embryos in culturesuggesting that it may not be mutagenic nor highly toxic (Teratology 39,31-37 (1989)).

Since mutagenicity and toxicity are not highly predictable properties,non-mutagenic prodrugs of formula I and their associated by-products canbe readily identified by conducting well known in vitro and in vivoassays. For example, compounds can be tested in non-mammalian cellassays such as the Ames test, a fluctuation test in Kl. pneumoniae, aforward mutation assay with S. typhimurium, a chromosome loss assay inSaccharomyces cerevisiae, or a D3 recombinogenicity assay inSaccharomyces cerevisiae. Compounds can also be tested in mammalian cellassays such as the mouse lymphoma cells assay (TK+/−heterozygotes ofL5178Y mouse lymphoma cells), assays in Chinese hamster ovary cells(e.g. CHO/HGPRT assay), and an assay in rat liver cell lines (e.g. RL1or RL4). Each of these assays can be conducted in the presence ofactivators (e.g. liver microsomes) which may be of particular importanceto these prodrugs. By conducting these assays in the presence of theliver microsomes, for example, the prodrug produces products, such asphenol or vinyl ketone. The mutagenicity of the by-product is measuredeither directly or as a prodrug where the results are compared to theparent drug alone. Assays in liver cell lines are a preferred aspect ofthe invention since these cells have higher glutathione levels, whichcan protect the cell from damage caused by a Michael acceptor, as wellas greater levels of intracellular enzymes used to detoxify compounds.For example, the liver contains reductases that with some by-productsmight result in reduction of the carbonyl.

A variety of end points are monitored including cell growth, colonysize, gene mutations, micronuclei formation, mitotic chromosome loss,unscheduled DNA synthesis, DNA elongation, DNA breaks, morphologicaltransformations, and relative mitotic activity.

In vivo assays are also known that assess the mutagenicity andcarcinogenicity of compounds. For example, a non-mammalian in vivo assayis the Drosophila sex-linked recessive lethal assay. Examples ofmammalian in vivo assays include the rat bone marrow cytogenetic assay,a rat embryo assay, as well as animal teratology and carcinogenicityassays.

Kinase Bypass

Nucleosides are often converted in cells to mono-, di- andtri-phosphates with the latter usually acting as the biologically activespecies. In some cases, the triphosphate is a potent inhibitor of thetarget enzyme but poor to modest efficacy is achieved in vivo becauseadministration of the nucleoside fails to achieve sufficiently highlevels of the biologically active phosphate. Often the failure toachieve high levels of the phosphate is because the nucleoside is a poorsubstrate of the enzymes that catalyze the phosphorylation reaction.Other reasons include the low natural levels of the phosphorylatingenzyme in the target cell which may represent low natural expressionlevels or be a result of chronic therapy with the drug or another drugwhich results in enzyme down regulation. In some cases, drug resistanceresults from a decrease in activity of the enzymes responsible forsynthesis of a nucleoside monophosphate (e.g. kinases such asthymidylate kinase or enzymes in the biosynthesis pathway of5-fluoro-2′-deoxy UMP). In other cases nucleoside transporters aredownregulated leading to lower intracellular nucleoside drugconcentration and therefore less nucleoside is available forphosphorylation. Similarly, increased expression of multidrug resistantgene product is postulated to increase the export of nucleotides fromcancer cells. Administration of the prodrug generates the monophosphateby a different pathway avoiding the pathways that cause the resistanceto the parent drug. Thus, the prodrugs of the present invention canachieve a therapeutic effect in cells resistant to the parent drug. Somedrugs as the mono- or triphosphate analogs are highly potent inhibitorsof the target enzyme (e.g. HBV polymerase) but are poorly effective incells or in vivo due to poor phosphorylation. (Example H) These drugsare especially preferred drugs since the prodrug strategy delivers themonophosphate. Frequently, the first phosphorylation of the nucleosideis the rate-limiting step whereas phosphorylation of the monophosphateto the triphosphate by mammalian kinases is rapid and relativelyinsensitive to structural variations.

Ring-Substituted Cyclophosphamide Analogs for Treatment of CertainCancers:

Cyclophosphamide (CPA) and Ifosfamide (IF) are commonly used oncolyticdrugs that undergo initial activation in the liver via a P450 mechanism(CYP2B6 and CYP3A4) to intermediate 7. This intermediate exists inequilibrium with the ring-opened species (8), which is the penultimateintermediate for a variety of metabolic pathways, including aβ-elimination reaction that generates the alkylating mustard 9 andacrolein. The latter reaction sequence occurs via a non-enzymaticreaction and is thought to proceed with a t½ of ˜30-40 minutes atneutral pH.

Intermediates 8 and 9 are relatively lipophilic and long-lived. As aconsequence, a significant proportion of the activated CPA “escapes” theliver and enters the general circulation. Both the oncolytic activityand the drug-associated side effects are due to extra-hepatic breakdown.Uptake of the intermediate by tumor cells and generation of the mustardresults in inhibition of cell proliferation. Similarly, myelosuppressionand decreased white cells are side effects associated with CPA therapythat result from inhibition of extra-hepatic cell proliferation.

CPA and IF are under investigation in conjunction with methods usefulfor the introduction of cytochrome P450s artificially, e.g. byretrovirus and other well known gene therapy strategies as well as viaimplanted cells engineered to express cytochrome P450s for the treatmentof cancer. The hope is that with cells that express the CYP near thetumor, a more local and therefore a potentially more beneficial therapyis possible. Prodrugs of this invention have the advantage for treatingprimary and secondary liver cancers as well as for tumors with enhancedP450 activity since they generate the active oncolytic agent morelocally, i.e. with less “escape” from the tissue containing the P450activity. The reasons for the greater local effect are due todifferences in the prodrug cleavage intermediates which include: (1) aring-opening reaction that is rapid and irreversible; and (2) thering-opened product, which can be negatively charged (M-P(O)(NHR⁶)O⁻)and therefore unable to exit the cell via passive diffusion.

Types of Parent Drugs

Various kinds of parents drugs can benefit from the prodrug methodologyof the present invention. Parent drugs of the form MH, which arephosphorylated to become the biologically active drug are well suitedfor use in the prodrug methodology of the present invention. There aremany well known parent drugs of the form MH which become biologicallyactive via phosphorylation. For example, it is well known that antitumorand antiviral nucleosides are activated through phosphorylation. Thesecompounds include LdC, LdT, araA; AZT; d4T; ddI; ddA; ddC; L-ddC; LFddC; L-d4C; L-Fd4C; 3TC; ribavirin; 5-fluoro-2′-deoxyuridine; FIAU;FIAC; BHCG; L FMAU; BvaraU; E-5-(2-bromovinyl-2′ deoxyuridine; TFT;5-propynyl-1 arabinosyluracil; CDG; DAPD; FDOC; d4C; DXG; FEAU; FLG;FLT; FTC; 5-yl-carbocyclic 2′ deoxyguanosine; oxetanocin A; oxetanocinG; Cyclobut A, Cyclobut G; dFdC; araC; bromodeoxyuridine; IDU; CdA;FaraA; Coformycin, 2′-deoxycoformycin; araT; tiazofurin; ddAPR;9-(arabinofuranosyl)-2,6 diaminopurine; 9-(2′-deoxyribofuranosyl)-2,6diaminopurine; 9-(2′-deoxy 2′ fluororibofuranosyl)-2,6-diaminopurine; 9(arabinofuranosyl)guanine; 9-(2′ deoxyribofuranosyl)guanine; 9-(2′-deoxy2′fluororibofuranosyl)guanine; FMdC; 5,6 dihydro-5-azacytidine;5-azacytidine; 5-aza 2′deoxycytidine; AICAR; ACV, GCV; penciclovir;(R)-9-(3,4 dihydroxybutyl)guanine; cytallene; PMEA, PMEDAP, HPMPC,HPMPA, FPMPA, and PMPA.

In one preferred aspect, the compounds of formula I does not includecompounds where MH is araA or 5-fluoro-2′-deoxyuridine.

Preferred antiviral drugs include:

LdC, LdT, araA; AZT; d4T; ddI; ddA; ddC; L-ddC; L FddC; L-d4C; L-Fd4C;3TC; ribavirin; FIAU; FIAC; BHCG; L-FMAU; BvaraU; E-5-(2bromovinyl-2′-deoxyuridine; TFT; 5-propynyl 1-arabinosyluracil; CDG;DAPD; FDOC; d4C; DXG; FEAU; FLG; FLT; FTC; 5-yl-carbocyclic2′deoxyguanosine; cytallene; oxetanocin A; oxetanocin G; Cyclobut A,Cyclobut G; bromodeoxyuridine; IDU; araT; tiazofurin; ddAPR;9-(arabinofuranosyl)-2,6 diaminopurine; 9-(2′-deoxyribofuranosyl)-2,6diaminopurine; 9-(2′-deoxy 2′ fluororibofuranosyl)-2,6-diaminopurine; 9(arabinofuranosyl)guanine; 9-(2′deoxyribofuranosyl)guanine; 9-(2′-deoxy2′ fluororibofuranosyl)guanine; FMdC; 5,6 dihydro-5-azacytidine;5-azacytidine; 5-aza 2′deoxycytidine; ACV, GCV; penciclovir; (R)9-(3,4-dihydroxybutyl)guanine; cytallene; 5 PMEA; PMEDAP; HPMPC; HPMPA;FPMPA; PMPA; foscarnet; and phosphonoformic acid.

In one preferred aspect, MH does not include araA.

More preferred antiviral drugs include:

araA; AZT; d4T; ddI; ddA; ddC; L-ddC; L FddC; L-d4C; L-Fd4C; 3TC;ribavirin; FIAU; FIAC; L-FMAU; TFT; CDG; DAPD; FDOC; d4C; DXG; FEAU;FLG; FLT; FTC; 5-yl carbocyclic 2′ deoxyguanosine; cytallene; oxetanocinA; oxetanocin G; Cyclobut A, Cyclobut G; araT; ACV, GCV; penciclovir;PMEA; PMEDAP; HPMPC; HPMPA; PMPA; foscarnet.

In one preferred aspect, MH does not include araA.

Most preferred antiviral drugs include:

3TC;

penciclovir;

FMAU;

DAPD;

FTC;

Cyclobut G;

ACV;

GCV;

PMEA;

HPMPA;

5-yl-carbocyclic 2′ deoxyguanosine;

ribavirin

Preferred anticancer drugs include:

dFdC; 2′,2′-difluorodeoxycytidine (gemcitabine);

araC; arabinosylcytosine (cytarabine);

F-ara-A; 2-fluoroarabinosyladenosine (fludarabine); and

CdA; 2-chlorodeoxyadenosine (cladribine).

2′-deoxy-5-iodouridine

Coformycin

2′-deoxycoformycin

Tiazofurin

Ribavirin

5-fluoro-2′deoxyuridine

9-(arabinofuranosyl)-2,6-diaminopurine

9-(2′-deoxyribofuranosyl)-2,6-diaminopurine

9-(2′-deoxy-2′-fluororibofuranosyl)-2,6-diaminopurine

9-(arabinofuranosyl)-guanine

9-(2′-deoxyribofuranosyl)-guanine

9-(2′-deoxy-2′-fluororibofuranosyl)-guanine

In one preferred aspect, MH does not include 5-fluoro-2′-deoxyuridine.

In one aspect, preferably the compounds of formula I are not used in thetreatment of diabetes.

More preferred anticancer drugs include:

dFdC;

araC;

FaraA;

CdA;

5-fluoro 2′deoxyuridine;

GCV;

5,6-dihydro-5-azacytidine;

5-azacytidine; and

5-aza-2′-deoxycytidine.

In one preferred aspect, ME does not include 5-fluoro-2′-deoxyuridine.

Drugs containing a phosphonic acid (C—PO₃ ²⁻) moiety are also suitableparent drugs advantageously used in the present invention. These drugsare biologically active either in the form of MPO₃ ²⁻, MP₂O₆ ³⁻, orMP₃O₉ ⁴⁻. Phosphonic acids that are also suitable for this prodrugdelivery strategy include protease inhibitors that are useful forexample as antihypertensives, anticancer or anti-inflammatory agents.The novel prodrug methodology can be applied to NEP inhibitors,(DeLambert et al., J. Med. Chem. 37:498 (1994)), ACE inhibitors,endothelin converting enzyme inhibitors, purine nucleoside phosphorylaseinhibitors, inhibitors of metalloproteases involved in tumor metastasis,and inhibitors of collagenase (Bird et al., J. Med. Chem. 37, 158-169(1994). Moreover, phosphonic acids useful as NMDA antagonists which areuseful for treating a variety of conditions, including stroke, headtrauma, pain, and epilepsy. Other phosphonic acids that could benefitfrom the prodrug strategies are phosphonic acids reported by Squibb thatinhibit squalene synthase, by Hoechst that are immunomodulators, byMerck that are antidepressants, by Ciba-Geigy and Marion Merrel Dow thatare immunosuppressants via inhibition of purine nucleosidephosphorylase, and by Bristol-Myers Squibb, Gilead that are antivirals.Certain antibiotics might be suitable, especially antibiotics such asD-alanine racemase inhibitors and fosfomycin and associated analogs.

The following compounds and their analogs can be used in the prodrugmethodology of the present invention:

NEP Inhibitors

-   (S)-3-[N-[2-[(phosphonomethyl)amino]-3-(4-biphenylyl)propionyl]amino]propionic    acid by DeLombaert et al. in J Med. Chem. 1994 Feb. 18;    37(4):498-511    Collagenase Inhibitors-   N,[N—((R)-1-phosphonopropyl(-(S)-leucyl]-(S)-phenylalanine N-methyl    amide by Bird et al. in J Med Chem 1994 Jan. 7; 37(1):158-69    Angiotensin Coverting Enzyme Inhibitors-   (IR)-1-(N—(N-acetyl-L-isoleucyl)-L-tyrosyl)amino-2-(4-hydroxyphenyl)ethyl-phosphonic    acid by Hirayama et al. in Int J Pept Protein Res 1991 July;    38(1):20-4.    Endothelin Inhibitor-   CGS 26303 by DeLombaert et al., Biochem Biophys Res Commun 1994 Oct.    14; 204(1):407-12-   (S,S)-3-Cyclohexyl-2-[[5-(2,4-difluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]amino]propionic    acid by Wallace et al., J Med Chem 1998 Apr. 23; 41(9):1513-23-   (S,S)-2-[[5-(2-fluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl-]amino]-4-methylpentanoic    acid-   (S,S)-2-[[5-(3-fluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]+++amino]-4-methylpentanoic    acid    NMDA/AMPA Antagonists-   N-phosphonoalkyl-5-aminomethylquinoxaline-2,3-diones as described in    Bioorg Med. Chem. Lett. 1999 Jan. 18; 9(2):249-54-   3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid by Bespalov    et al. in Eur J Pharmacol 1998 Jun. 26; 351(3):299-305-   [2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-2-yl)-ethyl]phosphonic    acid D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid-   6,7-dichloro-2(1H)-oxoquinoline-3-phosphonic acid by Desos et al.    in J. Med. Chem. 1996 Jan. 5; 39(1):197-206.-   cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS19755)    Purine Nucleoside Phosphorylase Inhibitors-   [7-(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)-1,1-fluoroheptyl]phosphonic    acid and-   [4-(5-amino-6,7-dihydro-7-oxo-3H-1,2,3,-triazolo[4,5-d]-pyrimidin-3-yl)butyl]phosphonic    acid.-   [[[5-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)pentyl]phosphinico]methyl]phosphonic    acid by Kelly et al. in J Med Chem 1995 Mar. 17; 38(6):1005-14-   (2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]-phenyl]ethenyl)-phosphonic    acid by Weibel et al. in Biochem Pharmacol. 1994 Jul. 19;    48(2):245-52.-   9-(3,3-Dimethyl-5-phosphonopentyl)guanine by Guida et al. in J Med    Chem 1994 Apr. 15; 37(8):1109-14.    Alanine Racemase Inhibitors-   DL-(1-Amino-2-propenyl)phosphonic acid by Vo-Quang et al. in J Med    Chem 1986 April; 29(4):579-81    Squalene Synthase Inhibitors-   1-Hydroxy-3-(methylpentylamino)-propylidene-1,1-bisphosphonic acid    by Amin et al. in Arzneimittelforschung. 1996 August; 46(8):759-62.-   BMS188494 is POM prodrug of BMS187745 by Dickson et al. in J Med.    Chem. 1996 Feb. 2; 39(3):661-4.    Treatment of Cancer:

The prodrug strategy in the current invention encompasses severalfeatures that are advantageously used in cancer therapies. Many of theknown anticancer drugs are nucleosides that undergo phosphorylation tothe monophosphate which frequently is further converted to thetriphosphate. The prodrug strategy increases the effectiveness of thesedrugs because the prodrug is cleaved by liver-abundant enzymes andtherefore higher levels of the active metabolite are achieved in theliver relative to extrahepatic tissues. The net effect is greaterefficacy and/or a greater therapeutic index. In some cases, the prodrugstrategy also increases the effectiveness of these drugs by bypassingwell-known resistance mechanisms including mechanisms involved in theuptake, intracellular biosynthesis of the active metabolite, export andmetabolism of the monophosphate. Examples of preferred drug candidatesthat are specifically amenable to the strategy include, e.g. dFdC, araC,F-ara, and CdA, and 5-fluoro-2′-deoxyuridine (“5-FU”). In one aspect, MHis not 5-FU.

Some prodrugs may result in some accumulation of the monophosphate incells. Certain monophosphates are useful for the treatment of cancers,e.g. monophosphates that are potent inhibitors of thymidylate synthase(TS) and IMP dehydrogenase. Some TS inhibitors are reported to bemoderately effective in treating liver cancers. For example, 5-FU and5-FdUMP are effective. These drugs, however, are plagued by drugresistance and severe side effects. To avoid the latter, 5-FU analogsare often delivered via the portal artery to achieve the highestpossible liver levels. Accordingly, 5-FdTMP and associated analogs aresuitable targets for the prodrug strategy. Other nucleosides such asribavirin, tiazofurin and coformycin and analogs thereof are alsosuitable targets for the prodrug strategy.

Liver cancers are also very resistant to radiotherapy. One method forenhancing radiotherapy is to administer radiosensitizers.Radiosensitizers such as 2′-deoxy-5-iodouridine are nucleosides suitablefor the prodrug technology since the prodrugs will be cleaved by onlyP450 containing cells. After cleavage the monophosphate can be furtherphosphorylated to the triphosphate and trapped inside the cell makingthe cell more sensitive to radiotherapy.

Treatment of Viral Infections:

Drugs useful for treating viruses that infect the liver and cause liverdamage, e.g. hepatitis virus strains, exhibit similar properties to theanticancer nucleoside drugs in terms of efficacy, side effects andresistance. Prodrugs of these nucleosides would therefore be useful intreating hepatitis. In some cases, the drugs are already targeted forhepatitis (e.g. araA, 3TC, L-FMAU, FTC, BMS 200,475). The prodrugs ofthese compounds could enhance the efficacy, increase the therapeuticindex, improve the pharmacodynamic half-life and/or bypass drugresistance. Prodrugs of other agents used to treat viral infectionsother than hepatitis may also be made useful by administration of theprodrugs of this invention since these drugs are good antivirals (e.g.acyclovir, but useful for other viral infections because they arephosphorylated to the monophosphono by a viral kinase. The monophosphateis converted to the biologically active triphosphate by mammaliankinases. Accordingly, delivery of the monophosphate using this clan ofprodrugs enables treatment of hepatitis by drugs normally used to treatother viral infections.

Use for the Delivery of Diagnostic Agents:

Nucleoside diagnostic agents, e.g. uridine analogs with the 5Hsubstituted with Tc, are useful as the prodrugs of this invention asliver diagnostic agents. These compounds are converted first to themonophosphate and then onto the triphosphate in cells that contain P450activity, specifically CYP3A4 activity. Since nearly all of the activityis in the liver, diagnostic agents of this type will primarily bemetabolized in the liver and accumulate in cells that metabolize theprodrug. Accordingly, liver tumors that have no CYP3A4 activity ortumors such as hepatocellular carcinomas which have approximately 50% ofthe normal activity may be differentiated from normal liver tissue.

Agents Used to Modulate CYP Activity

A variety of methods may be used to enhance the in vivo activity ofcompounds of formula I. For example, various drugs are known thatenhance cytochrome P450 (CYP) activity. Enhancement frequently entailsincreased gene transcription. Four families of CYPs are particularlysusceptible to induction, namely CYP1-4. Induction is purportedly viareceptors that are activated by various xenobiotics. For example, CYP1gene activation frequently involves activation of the Ah receptor bypolycyclic aromatic hydrocarbons. CYP2-4 are activated via orphannuclear receptors. Data suggests that the nuclear receptor CAR(constitutively Active Receptor) is responsible for phenobarbital CYPactivation, especially CYP2 genes. The pregnane nuclear receptors (PXRor PAR or SXR) are thought to activate CYP3A genes whereas the PPAR(peroxisome proliferator activate receptor) is linked to CYP4 geneactivation. All three xenobiotic receptors are highly expressed in theliver which accounts for the liver specificity of the P450 geneinduction.

Xenobiotics known to induce CYP3 genes include phenobarbital, a varietyof steroids, e.g. dexamethasone, antibiotics, e.g. rifampicin, andcompounds such as pregnenolone-16a carbonitrile, phenyloin,carbamazepine, phenylbutazone, etc. A variety of methods are known thatenable identification of xenobiotics that induce P450s, including areporter gene assay in HepG2 cells (Ogg et al., Xenobiotica 29, 269-279(1999). Other inducers of the CYP3A subfamily are known that act at thepost-transcriptional level either by mRNA or protein stabilization, e.g.clotrimazole, TA and erythromycin. Compounds known to induce CYPs oridentified in in vitro assays are then used to enhance CYP activity invivo. For example, CYP activity is monitored in rats pre-treated withCYP modulators by e.g. evaluating liver microsomes over a period of timeto determine the optimal pre-treatment period, dose and dosingfrequency. Rats with enhanced CYP activity, especially the CYP activityresponsible for activation of the prodrugs (e.g. CYP3A4), are thentreated with compounds of formula I. Enhanced CYP activity can then leadto enhanced prodrug conversion and liver specificity. For example,enhanced metabolism of cyclophosphamide was found with pre-treatmentwith phenobarbital (Yu et al., J. Pharm. Exp. Ther. 288, 928-937 (1999).

In some cases, enhanced CYP activity may lead to unwanted drugmetabolism. For example, enhanced activity of CYPs not involved inprodrug activation can result in increased drug metabolism and thereforedecreased efficacy. In addition, increased CYP activity in othertissues, e.g. CYP3A4 in the gastrointestinal tract, could result indecreased prodrug absorption and liver drug levels Inhibitors of CYPactivity are known that might be useful in minimizing unwanted drugmetabolism. For example, grapefruit juice is known to inactivategastrointestinal CYP3A4 and to result in enhanced absorption of numerousdrugs metabolized by CYP3A4. CYP inhibitors are also known for many ofthe CYP subfamilies that can be useful for attenuating unwanted drugmetabolism while maintaining CYP activity important for prodrugcleavage. For example, the CYP3A inhibitor TAO was used to modulatecyclophosphamide metabolism in vivo in a manner that decreased theformation of toxic metabolites that do not contribute to its antitumoractivity.

Methods for Monitoring Patient P450 Activity

CYP activity is known to exhibit significant differences acrossindividuals. The range for CYP3A4 is 5- to 20-fold although mostindividuals are within a 3-fold range. Modest decreases are noted forindividuals with liver disease (30-50%) or advanced age (25-50%).Differences for gender are even more modest (<25%). Methods forphenotyping an individual's CYP activity are known and could be usefulin predicting who should receive drugs that modulate CYP activity.Evasive procedures include liver biopsy. Non evasive procedures havebeen reported, including an “erythromycin breath test” which is based onthe exhalation of 14CO2 generated from the CYP3A-mediatedN-demethylation of radiolabeled erythromycin (iv). (Watkins,Pharmacogenetics 4, 171-184 (1994)).

Gene Therapy

Introduction into tumor cells genes that encode for enzymes not normallyexpressed represents a new therapeutic strategy for increasing thetherapeutic effectiveness of anticancer chemotherapies. The generalstrategy entails expression of an enzyme that catalyzes the breakdown ofa prodrug of an anticancer drug thereby localizing the drug in or nearthe tumor mass and limiting exposure elsewhere. The strategy has beendemonstrated using the HSV-TK gene wherein the thymidylate kinasespecifically expressed in the transfected cells activates ganciclovir tothe monophosphate which is then converted by other kinases to the tumorcell killing triphosphate. A similar strategy uses the bacterialcytosine deaminase gene for conversion of 5-fluorouracil to5-fluorocytosine. Other genes have been considered includingcarboxypeptidase G2, nitro reductase, purine nucleoside phosphorylation,etc. In addition, CYP gene transfer has been explored as a way toenhance the chemotherapeutic effect of cyclophosphamide and ifosfamide,two drugs known to be activated by CYPs. For example, human breastcancer cells were sensitized by transfection with the CYP2B1 gene (Chenet al., Cancer Research, 56, 1331 1340 (1996)). The advantage of thisstrategy relative to the HSV-TK gene strategy is that the product of theCYP catalyzed oxidation readily diffuses outside of the tumor cell andinto nearby cells. In contrast to monophosphate products of the HSV-TKstrategy, the product can enter cells that are not in cell-cell contactand therefore produce a more widespread tumor killing effect (Chen andWaxman, Cancer Research, 55, 581-589 (1995)).

Compounds of formula I can be made more effective by using gene therapyto introduce the gene that encodes the CYP specifically involved inprodrug cleavage. The specific CYP that breaks down the prodrug isreadily determined using some or all of the following steps: 1)demonstrate prodrug cleavage using human microsomes; 2) classify thesubfamily by comparing activity with microsomes induced with varioussubfamily specific inducers (e.g. CYP3 enzymes are induced with avariety of steroids, e.g. dexamethasone, antibiotics, e.g. rifampicin,and compounds such as pregnenolone-16a carbonitrile, phenyloin,carbamazepine, phenylbutazone, etc.; 3) identify the CYP or CYPsresponsible for prodrug activation by using known CYP subfamily specificinhibitors (e.g. troleandomycin, erythromycin, ketoconazole andgestodene) and/or by using neutralizing antibodies; 4) confirm CYPsubfamily by demonstrating turnover via the recombinant enzyme.

Genes are introduced to the tumor using a suitable vector (e.g.retroviral vectors, adenoviral vectors) or via direct DNA injection.P450 activity can also be introduced into or near the tumor mass usingcells engineered to express P450s. Preferred are encapsulated cells(e.g. Lohr et al., Gene Therapy 5: 1070 (1998)) The compounds of formulaI are then introduced following significant enhancement of the CYPactivity in the tumor.

Especially preferred are those prodrugs disclosed in the invention thatare converted to the parent phosph(on)ate in cells and tissues,especially hepatocytes and liver, as indicated by measurement of theintracellular drug metabolites in hepatocytes using the proceduredescribed in Examples G and J.

Preferred Compounds

The compounds of the invention are substituted 6-membered cyclic 1,3propane prodrugs of certain phosph(on)ates as represented by Formula I:

wherein:

V, W, and W′ are independently selected from the group consisting of —H,alkyl, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl,substituted heteroaryl, 1-alkenyl, and 1-alkynyl; or

together V and Z are connected via an additional 3-5 atoms to form acyclic group containing 5-7 ring atoms, optionally 1 heteroatom,substituted with hydroxy, acyloxy, alkoxycarbonyloxy, oraryloxycarbonyloxy attached to a carbon atom that is three atoms fromboth Y groups attached to the phosphorus; or

together V and Z are connected via an additional 3-5 atoms to form acyclic group, optionally containing 1 heteroatom, said cyclic group isfused to an aryl group at the beta and gamma position to the Y adjacentto V;

together V and W are connected via an additional 3 carbon atoms to forman optionally substituted cyclic group containing 6 carbon atoms andsubstituted with one substituent selected from the group consisting ofhydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, andaryloxycarbonyloxy, attached to one of said additional carbon atoms thatis three atoms from a Y attached to the phosphorus;

together Z and W are connected via an additional 3-5 atoms to form acyclic group, optionally containing one heteroatom, and V must be aryl,substituted aryl, heteroaryl, or substituted heteroaryl;

together W and W′ are connected via an additional 2-5 atoms to form acyclic group, optionally containing 0-2 heteroatoms, and V must be aryl,substituted aryl, heteroaryl, or substituted heteroaryl;

Z is selected from the group consisting of —CHR²OH, —CHR²OC(O)R³,—CHR²OC(S)R³, —CHR²OC(S)OR³, —CHR²OC(O)SR³, —CHR²OCO₂R, —OR², —SR²,CHR²N₃, —CH₂aryl, —CH(aryl)OH, —CH(CH═CR² ₂)OH, —CH(C≡CR²)OH, —R, —NR²₂, —OCOR³, —OCO₂R³, —SCOR³, —SCO₂R³, —NHCOR², —NHCO₂R³, —CH₂NHaryl,—(CH₂)_(p-)OR¹², and —(CH₂)_(p-)SR¹²;

p is an integer 2 or 3;

with the provisos that:

a) V, Z, W, W′ are not all —H; and

b) when Z is —R², then at least one of V, W, and W′ is not —H, alkyl,aralkyl, or alicyclic;

R² is selected from the group consisting of R³ and —H;

R³ is selected from the group consisting of alkyl, aryl, alicyclic, andaralkyl;

R⁶ is selected from the group consisting of —H, and lower alkyl,acyloxyalkyl, alkoxycarbonyloxy alkyl and lower acyl;

R¹² is selected from the group consisting of —H, and lower acyl;

each Y is independently selected from the group consisting of —O—, —NR⁶—with the proviso that at least one Y is —NR⁶—;

M is selected from the group that attached to PO₃ ²⁻, P₂O₆ ³, P₃O₉ ⁴⁻ orP(O)(NHR⁶)O⁻ is a biologically active agent, but is not an FBPaseinhibitor, and is attached to the phosphorus in formula I via a carbon,oxygen, sulfur or nitrogen atom;

with the provisos that:

1) M is not —NH(lower alkyl), —N(lower alkyl)₂, —NH(lower alkylhalide),—N(lower alkylhalide)₂, or —N(lower alkyl) (lower alkylhalide); and

2) R⁶ is not lower alkylhalide;

and pharmaceutically acceptable prodrugs and salts thereof.

In general, preferred substituents, V, Z, W, and W′ of formula I arechosen such that they exhibit one or more of the following properties:

(1) enhance the oxidation reaction since this reaction is likely to bethe rate determining step and therefore must compete with drugelimination processes.

(2) enhance stability in aqueous solution and in the presence of othernon-P450 enzymes;

(3) enhance cell penetration, e.g. substituents are not charged or ofhigh molecular weight since both properties can limit oralbioavailability as well as cell penetration;

(4) promote the β-elimination reaction following the initial oxidationby producing ring-opened products that have one or more of the followingproperties:

-   -   a) fail to recyclize;    -   b) undergo limited covalent hydration;    -   c) promote β-elimination by assisting in the proton abstraction;    -   d) impede addition reactions that form stable adducts, e.g.        thiols to the initial hydroxylated product or nucleophilic        addition to the carbonyl generated after ring opening; and    -   e) limit metabolism of reaction intermediates (e.g. ring-opened        ketone);

(5) lead to a non-toxic and non-mutagenic by-product with one or more ofthe following characteristics. Both properties can be minimized by usingsubstituents that limit Michael additions, e.g.:

-   -   a) electron donating Z groups that decrease double bond        polarization;    -   b) W groups that sterically block nucleophilic addition to the        β-carbon;    -   c) Z groups that eliminate the double bond after the elimination        reaction either through retautomerization (enol->keto) or        hydrolysis (e.g. enamine);    -   d) V groups that contain groups that add to the α,β-unsaturated        ketone to form a ring;    -   e) Z groups that form a stable ring via Michael addition to        double bond; and    -   f) groups that enhance detoxification of the by-product by one        or more of the following characteristics:        -   (i) confine to liver; and        -   (ii) make susceptible to detoxification reactions (e.g.            ketone reduction); and

(6) capable of generating a pharmacologically active product.

Suitable alkyl groups include groups having from 1 to about 20 carbonatoms. Suitable aryl groups include groups having from 1 to about 20carbon atoms. Suitable aralkyl groups include groups having from 2 toabout 21 carbon atoms. Suitable acyloxy groups include groups havingfrom 1 to about 20 carbon atoms. Suitable alkylene groups include groupshaving from 1 to about 20 carbon atoms. Suitable alicyclic groupsinclude groups having 3 to about 20 carbon atoms. Suitable heteroarylgroups include groups having from 1 to about 20 carbon atoms and from 1to 4 heteroatoms, preferably independently selected from nitrogen,oxygen, and sulfur. Suitable heteroaliacyclic groups include groupshaving from 2 to about twenty carbon atoms and from 1 to 5 heteroatoms,preferably independently selected from nitrogen, oxygen, phosphorous,and sulfur.

In compounds of formula I, preferably one Y is —O— and one Y is —NR⁶—.When only one Y is —NR⁶—, preferably the Y closest to W and W′ is —O—.In another aspect, preferably the Y closest to V is —O—.

In another aspect, both Y groups are —NR⁶—.

More preferred are compounds wherein one Y is —O—, and

V, W, and W′ are independently selected from the group consisting of —H,alkyl, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl,substituted heteroaryl, 1-alkenyl, and 1-alkynyl, or

together V and W are connected via an additional 3 carbon atoms to forman optionally substituted cyclic group containing 6 carbon atoms andsubstituted with one substituent selected from the group consisting ofhydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, andaryloxycarbonyloxy, attached to one of said additional carbon atoms thatis three atoms from a Y attached to the phosphorus.

More preferred are such compounds where V is selected from the groupconsisting of aryl, substituted aryl, heteroaryl, and substitutedheteroaryl.

More preferred V groups of formula VI are aryl, substituted aryl,heteroaryl, and substituted heteroaryl. Preferably, one Y is —O—.Particularly preferred aryl and substituted aryl groups include phenyl,and phenyl substituted with 1-3 halogens. Especially preferred are3,5-dichlorophenyl, 3-bromo-4-fluorophenyl, 3-chlorophenyl, and3-bromophenyl.

It is also especially preferred when V is selected from the groupconsisting of monocyclic heteroaryl and monocyclic substitutedheteroaryl containing at least one nitrogen atom. Most preferred is whensuch heteroaryl and substituted heteroaryl is 4-pyridyl, and3-bromopyridyl, respectively.

In another particularly preferred aspect, one Y group is —O—, Z, W, andW′ are H, and V is phenyl substituted with 1-3 halogens. Especiallypreferred are 3,5-dichlorophenyl, 3-bromo-4-fluorophenyl,3-chlorophenyl, and 3-bromophenyl.

It is also especially preferred when V is selected from the groupconsisting of heteroaryl and substituted heteroaryl.

Most preferred is when such heteroaryl is 4-pyridyl.

In another aspect, it is preferred when together V and W are connectedvia an additional 3 carbon atoms to form an optionally substitutedcyclic group containing 6 carbon atoms and monosubstituted with onesubstituent selected from the group consisting of hydroxy, acyloxy,alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy attachedto one of said additional carbon atoms that is three atoms from a Yattached to the phosphorus. In such compounds, it is more preferred whentogether V and W form a cyclic group selected from the group consistingof —CH₂—CH(OH)—CH₂—, —CH₂CH(OCOR³)—CH₂—, and —CH₂CH(OCO₂)R³)—CH₂—.

Another preferred V group is 1-alkene. Oxidation by P450 enzymes isknown to occur at benzylic and allylic carbons.

In one aspect, preferred V groups include —H, when Z is —CHR²OH,—CH₂OCOR³, or —CH₂OCO₂R³.

In another aspect, when V is aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl, preferred Z groups include —OR², —SR², —R², —NR²₂, —OCOR³, —OCO₂R³, —SCOR³, —SCO₂R³, —NHCOR², —NHCO₂R³, —(CH₂)_(p-)OR²,and —(CH₂)_(p-)SR². More preferred Z groups include —OR², R², —OCOR²,—OCO₂R³, —NHCOR², —NHCO₂R³, —(CH₂)_(p-)OR¹², and —(CH₂)_(p-)SR¹². Mostpreferred Z groups include —OR², —H, —OCOR³, —OCO₂R³, and —NHCOR².

Preferred W and W′ groups include H, R³, aryl, substituted aryl,heteroaryl, and substituted aryl. Preferably, W and W′ are the samegroup. More preferred is when W and W′ are H.

In one aspect, prodrugs of formula VI which, in addition, are preferred:

wherein

V is selected from the group consisting of aryl, substituted aryl,heteroaryl, and substituted heteroaryl. More preferred are suchcompounds where M is attached to the phosphorus via an O or N atom.Especially preferred are 3,5-dichlorophenyl, 3-bromo-4-fluorophenyl,3-chlorophenyl, 3-bromophenyl. Preferably Y is —O—. Particularlypreferred aryl and substituted aryl groups include phenyl andsubstituted phenyl. Particularly preferred heteroaryl groups includemonocyclic substituted and unsubstituted heteroaryl groups. Especiallypreferred are 4-pyridyl and 3-bromopyridyl.

In one aspect, the compounds of formula VI preferably have a group Zwhich is H, alkyl, alicyclic, hydroxy, alkoxy, OC(O)R³, OC(O)OR³, orNHC(O)R². Preferred are such groups in which Z decreases the propensityof the by-product, vinylaryl ketone to undergo Michael additions.Preferred Z groups are groups that donate electrons to the vinyl groupwhich is a known strategy for decreasing the propensity ofα,β-unsaturated carbonyl compounds to undergo a Michael addition. Forexample, a methyl group in a similar position on acrylamide results inno mutagenic activity whereas the unsubstituted vinyl analog is highlymutagenic. Other groups could serve a similar function, e.g. Z=OR¹²,NHAc, etc. Other groups may also prevent the Michael addition especiallygroups that result in removal of the double bond altogether such asZ=—OH, —OC(O)R³, —OCO₂R³, and NH₂, which will rapidly undergoretautomerization after the elimination reaction. Certain W and W′groups are also advantageous in this role since the group(s) impede theaddition reaction to the β-carbon or destabilize the product. Anotherpreferred Z group is one that contains a nucleophilic group capable ofadding to the α,β-unsaturated double bond after the elimination reactioni.e. (CH₂)_(p)SH or (CH₂)_(p)OH where p is 2 or 3. Yet another preferredgroup is a group attached to V which is capable of adding to theα,β-unsaturated double bond after the elimination reaction:

In another aspect, prodrugs of formula VII are preferred:

wherein

Z is selected from the group consisting of:

—CHR²OH, —CHR²OC(O)R³, —CHR²OC(S)R³, —CHR²OCO₂R³, —CHR²OC(O)SR³,—CHR²OC(S)OR³, and —CH₂aryl. Preferably, M is attached to the phosphorusvia a nitrogen. More preferred groups include —CHR²OH, —CHR²OC(O)R³, and—CHR²OCO₂R³. Preferably R² is H.

In another aspect, prodrugs of formula VIII are preferred:

wherein

Z′ is selected from the group consisting of —OH, —OC(O)R³, OCO₂R³, and—OC(O)SR³;

D⁴ and D³ are independently selected from the group consisting of —H,alkyl, —OH, and —OC(O)R³; with the proviso that at least one of D⁴ andD³ are —H. Preferably Y is —O—. An especially preferred Z′ group is OH.

In one preferred embodiment, W′ and Z are —H, W and V are both the samearyl, substituted aryl, heteroaryl, or substituted heteroaryl, and bothY groups are the same —NR⁶—, such that the phosphonate prodrug moiety:

has a plane of symmetry through the phosphorous-oxygen double bond.

In another preferred embodiment, W and W′ are H, V is selected from thegroup consisting of aryl, substituted aryl, heteroaryl, substitutedheteroaryl, and Z is selected from the group consisting of —H, OR², and—NHCOR². More preferred are such compounds where Z is —H. Preferably,such compound have M attached via oxygen. Most preferred are suchcompounds where oxygen is in a primary hydroxyl group. Also morepreferred, are those compounds where V is phenyl or substituted phenyl.

In one aspect, tumor cells expressing a P450 enzyme can be treated byadministering a cyclophosphamide analog selected from the groupconsisting of

wherein:

V, W, and W′ are independently selected from the group consisting of —H,alkyl, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl,substituted heteroaryl, 1-alkenyl, and 1-alkynyl; or

together V and Z are connected via an additional 3-5 atoms to form acyclic group containing 5-7 ring atoms, optionally 1 heteroatom,substituted with hydroxy, acyloxy, alkoxycarbonyloxy, oraryloxycarbonyloxy attached to a carbon atom that is three atoms fromboth Y groups attached to the phosphorus; or

together V and Z are connected via an additional 3-5 atoms to form acyclic group, optionally containing 1 heteroatom, said cyclic group isfused to an aryl group at the beta and gamma position to the Y adjacentto V;

together V and W are connected via an additional 3 carbon atoms to forman optionally substituted cyclic group containing 6 carbon atoms andsubstituted with one substituent selected from the group consisting ofhydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, andaryloxycarbonyloxy, attached to one of said additional carbon atoms thatis three atoms from a Y attached to the phosphorus;

together Z and W are connected via an additional 3-5 atoms to form acyclic group, optionally containing one heteroatom, and V must be aryl,substituted aryl, heteroaryl, or substituted heteroaryl;

together W and W′ are connected via an additional 2-5 atoms to form acyclic group, optionally containing 0-2 heteroatoms, and V must be aryl,substituted aryl, heteroaryl, or substituted heteroaryl;

Z is selected from the group consisting of —CHR²OH, —CHR²OC(O)R³,—CHR²OC(S)R³, —CHR²OC(S)OR³, —CHR²OC(O)SR³, —CHR²OCO₂R³, OR², —SR²,—CHR²N₃, —CH₂aryl, —CH(aryl)OH, —CH(CH═CR² ₂)OH, —CH(C≡CR²)OH, —R², NR²₂, —OCOR³, —OCO₂R³, —SCOR³, —SCO₂R³, —NHCOR², —NHCO₂R³, —CH₂NHaryl,—(CH₂)_(p-)OR¹², and —(CH₂)_(p-)SR¹²;

p is an integer 2 or 3;

with the provisos that:

a) V, Z, W, W′ are not all —H; and

b) when Z is —R², then at least one of V, W, and W′ is not —H, alkyl,aralkyl, or alicyclic;

R² is selected from the group consisting of R³ and —H;

R³ is selected from the group consisting of alkyl, aryl, alicyclic, andaralkyl;

R⁶⁶ is selected from the group consisting of —H, lower 2-haloalkyl, andlower alkyl;

R¹² is selected from the group consisting of —H, and lower acyl;

R″ is lower 2-haloalkyl;

R′″ is selected from the group consisting of H, lower alkyl, and R″;

each Y is independently selected from the group consisting of —O—,—NR⁶⁶— with the proviso that at least one Y is —NR⁶⁶—;

and pharmaceutically acceptable prodrugs and salts thereof.

Preferably, the tumor cell is a hepatocellular carcinoma. Thesecyclophosphamide analogs can also be used to treat tumor cells byenhancing the activity of a P450 enzyme that oxidizes cyclophosphamideanalogs and by administering the cyclophosphamide analogs.

In another aspect, the P450 activity is enhanced by the administrationof a compound that increases the amount of endogenous P450 enzyme.Preferably, the compound that increases the amount of endogenous P450enzyme is selected from the group consisting of phenobarbitol,dexamethasone, rifampicin, phentoin, and preganolon-16α-carbonitrile.Preferred cyclophosphoramide analogs include those where R″ is2-chloroethyl, and R′″ is selected from the group consisting of —H, and2-chloroethyl.

Also, preferred are cyclophosphamide analogs where R⁶⁶ is selected fromthe group consisting of —H, methyl, and 2-chloroethyl.

In one aspect, the activity of a P450 enzyme is enhanced byadministration of a compound that increases the amount of endogenousP450 enzyme.

Also preferred cyclophosphamide analogs are those where Z, W, W′, andR⁶⁶ are —H, and R″ and R′″ are 2-chloroethyl.

Preferred cyclophosphamide analogs have V is selected from the groupconsisting of aryl, substituted aryl, heteroaryl, and substitutedheteroaryl. More preferred are those compounds where Z, W, W′, and R⁶⁶are —H, and R″ and R′″ are 2-chloroethyl. Also more preferred are thosewherein V is selected from the group consisting of phenyl,3-chlorophenyl, and 3-bromophenyl. Also preferred are those wherein V is4-pyridyl.

Also more preferred, are those compounds where V is an optionallysubstituted monocyclic heteroaryl containing at least one nitrogen atom.Preferably such compounds have M attached via oxygen. Most preferred aresuch compounds where said oxygen is in a primary hydroxyl group.Especially preferred are such compounds where V is 4-pyridyl.

Preferably, oral bioavailability is at least 5%. More preferably, oralbioavailability is at least 10%.

P450 oxidation can be sensitive to stereochemistry which might either beat phosphorus or at the carbon bearing the aromatic group. The prodrugsof the present invention have two isomeric forms around the phosphorus.Preferred is the stereochemistry that enables both oxidation and theelimination reaction. Preferred is the cis-stereochemistry. In contrast,the reaction is relatively insensitive to the group M since cleavageoccurred with a variety of phosphonate, phosphate and phosphoramidates.The atom in M attached to phosphorus may be O, S or N. The active drugis M-PO₃ ²⁻, MP₂O₆ ³, or MP₃O₉ ⁴⁻ useful for treatment of diseases inwhich the liver is a target organ, including diabetes, hepatitis, livercancer, liver fibrosis, malaria and metabolic diseases where the liveris responsible for the overproduction of a biochemical end products suchas glucose (diabetes), cholesterol, fatty acids and triglycerides(atherosclerosis). Moreover, M-PO₃ ²⁻, MP₂O₆ ³⁻, or MP₃O₉ ⁴⁻ may beuseful in treating diseases where the target is outside the liver intissues or cells that can oxidize the prodrug.

Other preferred M groups include drugs useful in treating diabetes,cancer, viral infections, liver fibrosis, parasitic infections,hypertension, and hyperlipidemia.

The preferred compounds of formula VIII utilize a Z′ group that iscapable of undergoing an oxidative reaction that yields an unstableintermediate which via elimination reactions breaks down to thecorresponding M-P(O)(NHR⁶)₂, or M-P(O)(O⁻)(NHR⁶). An especiallypreferred Z′ group is OH. Groups D⁴ and D³ are preferably hydrogen,alkyl, —OR², —OCOR³, but at least one of D⁴ or D³ must be H.

The following compounds and their analogs can be used in the prodrugmethodology of the present invention.

In one preferred aspect, M is attached to the phosphorus in formula Ivia an oxygen atom. Preferably, M is a nucleoside. Preferably, M isattached via an oxygen that is in a primary hydroxyl group on aribofuranosyl or an arabinofuranosyl group. Preferably such compoundsinclude LdT, LdC, araA; AZT; d4T; ddI; ddA; ddC; L-ddC; L FddC; L-d4C;L-Fd4C; 3TC; ribavirin; 5-fluoro 2′ deoxyuridine; FIAU; FIAC; BHCG; LFMAU; BvaraU; E-5-(2-bromovinyl-2′ deoxyuridine; TFT; 5-propynyl-1arabinosyluracil; CDG; DAPD; FDOC; d4C; DXG; FEAU; FLG; FLT; FTC;5-yl-carbocyclic 2′ deoxyguanosine; oxetanocin A; oxetanocin G; CyclobutA, Cyclobut G; dFdC; araC; bromodeoxyuridine; IDU; CdA; FaraA;Coformycin, 2′-deoxycoformycin; araT; tiazofurin; ddAPR;9-(arabinofuranosyl)-2,6 diaminopurine; 9-(2′-deoxyribofuranosyl)-2,6diaminopurine; 9-(2′-deoxy 2′ fluororibofuranosyl)-2,6-diaminopurine; 9(arabinofuranosyl)guanine; 9-(2′ deoxyribofuranosyl)guanine; 9-(2′-deoxy2′fluororibofuranosyl)guanine; FMdC; 5,6 dihydro-5-azacytidine;5-azacytidine; 5-aza 2′deoxycytidine; or AICAR. In another aspect, it ispreferred when M is attached via an oxygen in a hydroxyl on an acyclicsugar it is preferred when such MH is ACV, GCV; penciclovir; (R)-9-(3,4dihydroxybutyl)guanine; or cytallene.

In one preferred aspect, the compounds in formula I do not includecompounds where MH is araA or 5-fluoro-2′-deoxyuridine.

In general, it is preferred that when M is attached via an oxygen, saidoxygen is in a primary hydroxy group. In such an instance, it ispreferred that MH is araA; AZT; d4T; ddI; ddA; ddC; L-ddC; L FddC;L-d4C; L-Fd4C; 3TC; ribavirin; 5-fluoro 2′ deoxyuridine; FIAU; FIAC;BHCG; L FMAU; BvaraU; E-5-(2-bromovinyl-2′ deoxyuridine; TFT;5-propynyl-1 arabinosyluracil; CDG; DAPD; FDOC; d4C; DXG; FEAU; FLG;FLT; FTC; 5-yl-carbocyclic 2′ deoxyguanosine; oxetanocin A; oxetanocinG; Cyclobut A, Cyclobut G; dFdC; araC; bromodeoxyuridine; IDU; CdA;FaraA; Coformycin, 2′-deoxycoformycin; araT; tiazofurin; ddAPR;9-(arabinofuranosyl)-2,6 diaminopurine; 9-(2′-deoxyribofuranosyl)-2,6diaminopurine; 9-(2′-deoxy 2′fluororibofuranosyl)-2,6-diaminopurine; 9(arabinofuranosyl)guanine; 9-(2′ deoxyribofuranosyl)guanine; 9-(2′-deoxy2′ fluororibofuranosyl)guanine; FMdC; 5,6 dihydro-5-azacytidine;5-azacytidine; 5-aza 2′deoxycytidine; AICAR; ACV, GCV; penciclovir;(R)-9-(3,4 dihydroxybutyl)guanine; or cytallene.

In another aspect, MH is araA.

In another aspect, MH is 5-fluoro-2′-deoxyuridine.

In another aspect, compounds of formula I wherein M is attached to thephosphorus in formula I via a carbon atom are preferred. In suchcompounds, preferably M-PO₃ ²⁻ is phosphonoformic acid, orphosphonoacetic acid.

In another preferred aspect, MPO₃ ²⁻, MP₂O₆ ³—, or MP₃O₉ ⁴⁻ is usefulfor the treatment of diseases of the liver or metabolic diseases wherethe liver is responsible for the overproduction of a biochemical endproduct. Preferably, such disease of the liver is selected from thegroup consisting of diabetes, hepatitis, cancer, fibrosis, malaria,gallstones, and chronic cholecystalithiasis. It is more preferred whentreating such diseases that MH, MPO₃ ²⁻, MP2O₆ ³, or MP₃O⁴⁻ is anantiviral or anticancer agent.

Preferably, the metabolic disease that MPO₃ ²⁻, MP₂O₆ ³⁻, or MP₃O₉ ⁴⁻are useful for diabetes, atherosclerosis, and obesity.

In another aspect, it is preferred when the biochemical end product isselected from the group consisting of glucose, cholesterol, fatty acids,and triglycerides. More preferred is when MP(O)(NHR⁶)O⁻ or MPO₃ ²⁻ is anAMP activated protein kinase activator.

In one preferred embodiment, W′ and Z are —H, W and V are both the samearyl, substituted aryl, heteroaryl, or substituted heteroaryl such thatthe phosphonate prodrug moiety:

has a plane of symmetry.

In another preferred embodiment, W and W′ are H, V is selected from thegroup consisting of aryl, substituted aryl, heteroaryl, substitutedheteroaryl, and Z is selected from the group consisting of —H, OR², and—NHCOR². More preferred are such compounds where Z is —H. Preferably,such compound have M attached via oxygen. Most preferred are suchcompounds where oxygen is in a primary hydroxyl group.

Also more preferred, are those compounds where V is phenyl orsubstituted phenyl. Most preferred are such compounds where said oxygenis in a primary hydroxyl group.

Preferably, such compounds have M attached via oxygen.

Also more preferred, are those compounds where V is an optionallysubstituted monocyclic heteroaryl containing at least one nitrogen atom.Preferably such compounds have M attached via oxygen. Most preferred aresuch compounds where said oxygen is in a primary hydroxyl group.

Especially preferred are such compounds where V is selected from thegroup consisting of phenyl substituted with 1-3 halogens, and 4-pyridyl.In these compounds it is also preferred when MH is selected from thegroup consisting of LdT, LdC, araA; AZT; d4T; ddI; ddA; ddC; L-ddC; LFddC; L-d4C; L-Fd4C; 3TC; ribavirin; 5-fluoro 2′ deoxyuridine; FIAU;FIAC; BHCG; L FMAU; BvaraU; E-5-(2-bromovinyl-2′deoxyuridine; TFT;5-propynyl-1 arabinosyluracil; CDG; DAPD; FDOC; d4C; DXG; FEAU; FLG;FLT; FTC; 5-yl-carbocyclic 2′deoxyguanosine; oxetanocin A; oxetanocin G;Cyclobut A, Cyclobut G; dFdC; araC; bromodeoxyuridine; IDU; CdA; FaraA;Coformycin, 2′-deoxycoformycin; araT; tiazofurin; ddAPR;9-(arabinofaranosyl)-2,6 diaminopurine; 9-(2′-deoxyribofuranosyl)-2,6diaminopurine; 9-(2′-deoxy 2′fluororibofuranosyl)-2,6-diaminopurine; 9(arabinofuranosyl)guanine; 9-(2′ deoxyribofuranosyl)guanine; 9-(2′-deoxy2′ fluororibofaranosyl)guanine; FMdC; 5,6 dihydro-5-azacytidine;5-azacytidine; 5-aza 2′ deoxycytidine; AICAR; ACV, GCV; penciclovir;(R)-9-(3,4 dihydroxybutyl)guanine; or cytallene. Particularly preferredare such compounds where V is selected from the group consisting ofphenyl substituted with 1-3 halogens and 4-pyridyl and MH is selectedfrom the group consisting of araA; AZT; d4T; 3TC; ribavirin; 5fluoro-2′deoxyuridine; FMAU; DAPD; FTC; 5-yl-carbocyclic2′deoxyguanosine; Cyclobut G; dFdC; araC; IDU; FaraA; ACV; GCV; orpenciclovir.

Also preferred is when MH is selected from the group consisting of ACV,GCV; penciclovir; (R)-9-(3,4 dihydroxybutyl)guanine; cytallene.

When W′ and W are H, V is aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl, and Z is H, OR², or —NHCOR², it is alsopreferred when M is attached to the phosphorus via a carbon atom.Preferred are such compounds wherein MPO₃ ²⁻ is selected from the groupconsisting of phosphonoformic acid, and phosphonoacetic acid. Alsopreferred are MH is selected from the group consisting of PMEA, PMEDAP,HPMPC, HPMPA, FPMPA, and PMPA.

Preferably, oral bioavailability is at least 5%. More preferably, oralbioavailability is at least 10%.

P450 oxidation can be sensitive to stereochemistry which might either beat phosphorus or at the carbon bearing the aromatic group. The prodrugsof the present invention have two isomeric forms around the phosphorus.Preferred is the stereochemistry that enables both oxidation and theelimination reaction. Preferred is the cis stereochemistry. In contrast,the reaction is relatively insensitive to the group M since cleavageoccurred with a variety of phosphonate, phosphate and phosphoramidates.Accordingly, the group M represents a group that as part of a compoundof formula I enables generation of a biologically active compound invivo via conversion to the corresponding M-PO₃ ²⁻. The atom in Mattached to phosphorus may be O, S, C or N. The active drug may be M-PO₃²⁻ or a metabolite of M-PO₃ ²⁻ such as the triphosphate useful fortreatment of diseases in which the liver is a target organ, includingdiabetes, hepatitis, liver cancer, liver fibrosis, malaria and metabolicdiseases where the liver is responsible for the overproduction of abiochemical end products such as glucose (diabetes), cholesterol, fattyacids and triglycerides (atherosclerosis). Moreover, M-PO₃ ²⁻ andMP(O)(NHR⁶)O⁻ may be useful in treating diseases where the target isoutside the liver but accessible to a phosph(on)ate. Preferred M groupsare groups in which M is a nucleoside and the phosphate is attached to ahydroxyl, preferably a primary hydroxyl on a sugar or sugar-analog. Morepreferred M groups include LdC, LdT, araA; AZT; d4T; ddI; ddA; ddC;L-ddC; L FddC; L-d4C; L-Fd4C; 3TC; ribavirin; 5-fluoro 2′deoxyuridine;FIAU; FIAC; BHCG; L FMAU; BvaraU; E-5-(2-bromovinyl-2′ deoxyuridine;TFT; 5-propynyl-1 arabinosyluracil; CDG; DAPD; FDOC; d4C; DXG; FEAU;FLG; FLT; FTC; 5-yl-carbocyclic 2′deoxyguanosine; oxetanocin A;oxetanocin G; Cyclobut A, Cyclobut G; dFdC; araC; bromodeoxyuridine;IDU; CdA; FaraA; Coformycin, 2′-deoxycoformycin; araT; tiazofurin;ddAPR; 9-(arabinofuranosyl)-2,6 diaminopurine;9-(2′-deoxyribofuranosyl)-2,6 diaminopurine; 9-(2′-deoxy 2′fluororibofuranosyl)-2,6-diaminopurine; 9 (arabinofuranosyl)guanine;9-(2′ deoxyribofuranosyl)guanine; 9-(2′-deoxy2′fluororibofuranosyl)guanine; FMdC; 5,6 dihydro-5-azacytidine;5-azacytidine; 5-aza 2′deoxycytidine; AICAR; ACV, GCV; penciclovir;(R)-9-(3,4 dihydroxybutyl)guanine; or cytallene. Especially preferredare lobucavir, FTC, 3TC, BMS 200,475; DAPD, DXG, L-FMAU, LdC, LdT,ribavirin, F-ara-A, araC, CdA, dFdC, 5-fluoro-2′-deoxyuridine, ACV, GCV,and penciclovir.

The preferred M groups include phosphonic acids useful for treatingviral infections. Such preferred antivirals include PMEA; PMEDAP; HPMPC;HPMPA; FPMPA; PMPA; foscarnet; phosphonoformic acid. More preferred arePMEA, PMPA, HPMPC, and HPMPA. Especially preferred are PMEA and PMPA.

Other preferred M groups include phosphonic acids useful in treatingdiabetes, liver fibrosis, e.g. collagenase inhibitors such as reportedin Bird et al., J. Med. Chem. 37, 158-169 (1994), parasitic infections,diseases responsive to metalloprotease inhibition (e.g. hypertension,liver, cancer), and hyperlipidemia.

In another aspect, preferred MH groups including L-nucleosides.Preferred L-nucleosides include FTC, 3TC, L-FMAU, LdC, and LdT.

In one aspect, preferred MH groups are acyclic nucleosides. Preferredacyclic nucleosides include ACV, GCV; penciclovir; (R)-9-(3,4dihydroxybutyl)guanine; and cytallene. More preferred are ACV; GCV, andpenciclovir.

In another aspect, preferred MH groups include dideoxy nucleosides.Preferred dideoxy nucleosides include AZT; d4T; ddI; ddA; ddC; L-ddC;L-FddC; L d4C; L-Fd4C; d4C; and ddAPR. More preferred are AZT; d4T; ddI;and ddC.

In another aspect, preferred MH groups include arabinofuranosylnucleosides. Preferred are araA; araT; 5-propynyl-1-arabinosyluracil;araC; FaraA; 9-(arabinofuranosyl)-2,6 diaminopurine; and9-(arabinofuranosyl)guanine. More preferred are araA; araC; and FaraA.

In another aspect, preferred MH groups include carbocyclic nucleosides.Preferred are 5-yl-carbocyclic 2′ deoxyguanosine; CDG; cyclobut A;cyclobut G; and BHCG. More preferred are 5-yl-carbocyclic 2′deoxyguanosine; and cyclobut G.

In another aspect, preferred MH groups include fluorinated sugars on thenucleosides. Preferred fluorinated sugars include FLT; FLG; FIAC; FIAU;FMAU; FEAU; dFdC; 9-(2′-deoxy-2′fluororibofuranosyl) 2,6-diaminopurine;and 9-(2′-deoxy 2′fluororibofuranosyl)guanine More preferred are L-FMAU;and dFdC.

In another aspect, preferred MH groups include dioxolane nucleosides.Preferred dioxolane nucleosides include DAPD; DXG; and FDOC. Morepreferred is DAPD and DXG.

In another aspect, preferred MH and MPO₃ ² for treating viral infectionsinclude lobucavir, FTC, 3TC, BMS 200,475; DAPD, DXG, L-FMAU, LdC, LdT,ribavirin, ACV, GCV, penciclovir, PMEA, and PMPA.

In another aspect, preferred MH groups for treating cancer includeF-ara-A, araC, CdA, dFdc, and 5-fluoro-2′-deoxyuridine.

The following prodrugs are preferred compounds of the invention. Thecompounds are shown without depiction of stereochemistry since thecompounds are biologically active as the diastereomeric mixture or as asingle stereoisomer. Compounds named in Table 1 are designated bynumbers assigned to the variables of formula VIa using the followingconvention:

M¹.V.L1.L2 where Y is NH and Y′ is oxygen; M¹ is a variable thatrepresents compounds of the formula M-H which have a specific hydroxylgroup that is phosphorylated with a group P(O)(Y—CH(V)CH2CH2-Y′) to makecompounds of formula VIa or M¹ is a variable that represents phosphonicacids of the formula M-PO₃ ²⁻ which are transformed to compounds offormula VIa by replacing two oxygens in the PO₃ ²⁻ group withY—CH(V)CH2CH2-Y′. V is an aryl or heteroaryl group that has 2substituents, L1 and L2, at the designated positions.

Variable M¹ is divided into three groups with the structures assigned toeach group listed below:

Variable M¹: Group M¹1:

-   -   1) 3TC where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.    -   2) (−)FTC where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl.    -   3) L-FMAU where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl.    -   4) Penciclovir where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl that is phosphorylated in cells.    -   5) BMS 200,475 where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl.    -   6) L(−)Fd4C where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl.    -   7) Lobucavir where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl that is phosphorylated in cells.    -   8) DXG where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.    -   9) LdC where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.        Variable M¹: Group M¹2:    -   1) ddI where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.    -   2) LdT where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.    -   3) ddC where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.    -   4) AZT where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.    -   5) d4T where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.    -   6) DAPD where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl.    -   7) L-FddC where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl.    -   8) Ribavirin where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl.    -   9) CdA where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the primary        hydroxyl        Variable M¹: Group M¹3:    -   1) Ganciclovir where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl that is phosphorylated in cells.    -   2) Acyclovir where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl.    -   3) Cytarabine where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl.    -   4) Gemcitabine where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl    -   5) Fludarabine where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to the        primary hydroxyl    -   6) Floxuridine where —P(O)(Y—CH(V)CH2CH2-Y′) is attached to        primary hydroxyl    -   7) HPMPC where —P(O)(Y—CH(V)CH2CH2-Y′) replaces the PO₃ ⁼ group.    -   8) PMEA where —P(O)(Y—CH(V)CH2CH2-Y′) replaces the PO₃ ⁼ group.    -   9) PMPA where —P(O)(Y—CH(V)CH2CH2-Y′) replaces the PO₃ ⁼ group.        Variable V: Group V1

1) 2-(L1)-3 (L2) phenyl

2) 2-(L1)-4(L2) phenyl

3) 2-(L1)-5(L2) phenyl

4) 2-(L1)-6(L2) phenyl

5) 3-(L1)-4(L2) phenyl

6) 3-(L1)-5(L2) phenyl

7) 3-(L1)-6(L2) phenyl

8) 2-(L1)-6(L2)-4-chlorophenyl

9) 3-(L1)-5 (L2) 4-chlorophenyl

Variable V: Group V2

1) 2-(L1)-3(L2) 4-pyridyl

2) 2-(L1)-5(L2) 4-pyridyl

3) 2-(L1)-6(L2) 4-pyridyl

4) 3-(L1)-5(L2) 4-pyridyl

5) 3-(L1)-6(L2) 4-pyridyl

6) 2-(L1)-4(L2) 3-pyridyl

7) 2-(L1)-5(L2) 3-pyridyl

8) 2-(L1)-6(L2) 3-pyridyl

9) 4-(L1)-5(L2) 3-pyridyl

Variable V: Group V3

1) 4-(L1)-6(L2) 3-pyridyl

2) 5-(L1)-6(L2) 3-pyridyl

3) 3-(L1)-4(L2) 2-pyridyl

4) 3-(L1)-5(L2) 2-pyridyl

5) 3-(L1)-6(L2) 2-pyridyl

6) 4-(L1)-5(L2) 2-pyridyl

7) 4-(L1)-6(L2) 2-pyridyl

8) 3-(L1)-4 (L2)-2-thienyl

9) 2-(L1)-5(L2) 3-furanyl

Variable L1

1) hydrogen

2) chloro

3) bromo

4) fluoro

5) methyl

6) isopropyl

7) methoxy

8) dimethylamino

9) acyloxy

Variable L2

1) hydrogen

2) chloro

3) bromo

4) fluoro

5) methyl

6) isopropyl

7) methoxy

8) dimethylamino

9) acyloxy

Preferred compounds are compounds listed in Table 1 using groups M¹1 andV1. For example, compound 1.3.6.7 represents structure 1 of group M¹1,i.e. 3TC; structure 3 of group V1, i.e. 2-(L1)-5-(L2) phenyl; structure6 of variable L1, i.e. isopropyl; and structure 7 of variable L2, i.e.methoxy. The compound 1.3.6.7. therefore is 3TC with theP(O)(Y—CH(V)CH2CH2Y′) attached to the primary hydroxyl group being{[1-(2-I-propyl-5-methoxyphenyl)-1,3-propyl]phosphoryl.

Preferred compounds are also compounds listed in Table 1 using groupsM¹1 and V2.

Preferred compounds are also compounds listed in Table 1 using groupsM¹1 and V3.

Preferred compounds are also compounds listed in Table 1 using groupsM¹2 and V1.

Preferred compounds are also compounds listed in Table 1 using groupsM¹2 and V2.

Preferred compounds are also compounds listed in Table 1 using groupsM¹2 and V3.

Preferred compounds are also compounds listed in Table 1 using groupsM¹3 and V1.

Preferred compounds are also compounds listed in Table 1 using groupsM¹3 and V2.

Preferred compounds are also compounds listed in Table 1 using groupsM¹3 and V3.

Preferred compounds are represented by all of the above named compoundswith the exception that Y is oxygen and Y′ is NH.

Preferred compounds are represented by all of the above named compoundswith the exception that Y and Y′ are NH.

Preferred compounds are represented by all of the above named compoundswith the exception that Y is NCH3 and Y′ is oxygen.

Preferred compounds are represented by all of the above named compoundswith the exception that Y is oxygen and Y′ is NCH₃.

TABLE 1 1.1.1.1. 1.1.1.2. 1.1.1.3. 1.1.1.4 1.1.1.5 1.1.1.6 1.1.1.71.1.1.8. 1.1.1.9 1.1.2.1 1.1.2.2 1.1.2.3 1.1.2.4 1.1.2.5 1.1.2.6 1.1.2.71.1.2.8 1.1.2.9 1.1.3.1 1.1.3.2 1.1.3.3 1.1.3.4 1.1.3.5 1.1.3.6 1.1.3.71.1.3.8 1.1.3.9 1.1.4.1 1.1.4.2 1.1.4.3 1.1.4.4 1.1.4.5 1.1.4.6 1.1.4.71.1.4.8 1.1.4.9 1.1.5.1 1.1.5.2 1.1.5.3 1.1.5.4 1.1.5.5 1.1.5.6 1.1.5.71.1.5.8 1.1.5.9 1.1.6.1 1.1.6.2 1.1.6.3 1.1.6.4 1.1.6.5 1.1.6.6 1.1.6.71.1.6.8 1.1.6.9 1.1.7.1 1.1.7.2 1.1.7.3 1.1.7.4 1.1.7.5 1.1.7.6 1.1.7.71.1.7.8 1.1.7.9 1.1.8.1 1.1.8.2 1.1.8.3 1.1.8.4 1.1.8.5 1.1.8.6 1.1.8.71.1.8.8 1.1.8.9 1.1.9.1 1.1.9.2 1.1.9.3 1.1.9.4 1.1.9.5 1.1.9.6 1.1.9.71.1.9.8 1.1.9.9 1.2.1.1 1.2.1.2 1.2.1.3 1.2.1.4 1.2.1.5 1.2.1.6 1.2.1.71.2.1.8 1.2.1.9 1.2.2.1 1.2.2.2 1.2.2.3 1.2.2.4 1.2.2.5 1.2.2.6 1.2.2.71.2.2.8 1.2.2.9 1.2.3.1 1.2.3.2 1.2.3.3 1.2.3.4 1.2.3.5 1.2.3.6 1.2.3.71.2.3.8 1.2.3.9 1.2.4.1 1.2.4.2 1.2.4.3 1.2.4.4 1.2.4.5 1.2.4.6 1.2.4.71.2.4.8 1.2.4.9 1.2.5.1 1.2.5.2 1.2.5.3 1.2.5.4 1.2.5.5 1.2.5.6 1.2.5.71.2.5.8 1.2.5.9 1.2.6.1 1.2.6.2 1.2.6.3 1.2.6.4 1.2.6.5 1.2.6.6 1.2.6.71.2.6.8 1.2.6.9 1.2.7.1 1.2.7.2 1.2.7.3 1.2.7.4 1.2.7.5 1.2.7.6 1.2.7.71.2.7.8 1.2.7.9 1.2.8.1 1.2.8.2 1.2.8.3 1.2.8.4 1.2.8.5 1.2.8.6 1.2.8.71.2.8.8 1.2.8.9 1.2.9.1 1.2.9.2 1.2.9.3 1.2.9.4 1.2.9.5 1.2.9.6 1.2.9.71.2.9.8 1.2.9.9 1.3.1.1 1.3.1.2 1.3.1.3 1.3.1.4 1.3.1.5 1.3.1.6 1.3.1.71.3.1.8 1.3.1.9 1.3.2.1 1.3.2.2 1.3.2.3 1.3.2.4 1.3.2.5 1.3.2.6 1.3.2.71.3.2.8 1.3.2.9 1.3.3.1 1.3.3.2. 1.3.3.3 1.3.3.4 1.3.3.5 1.3.3.6 1.3.3.71.3.3.8 1.3.3.9 1.3.4.1 1.3.4.2 1.3.4.3 1.3.4.4 1.3.4.5 1.3.4.6 1.3.4.71.3.4.8 1.3.4.9 1.3.5.1 1.3.5.2 1.3.5.3 1.3.5.4 1.3.5.5 1.3.5.6 1.3.5.71.3.5.8 1.3.5.9 1.3.6.1 1.3.6.2 1.3.6.3 1.3.6.4 1.3.6.5 1.3.6.6 1.3.6.71.3.6.8 1.3.6.9 1.3.7.1 1.3.7.2 1.3.7.3 1.3.7.4 1.3.7.5 1.3.7.6 1.3.7.71.3.7.8 1.3.7.9 1.3.8.1 1.3.8.2 1.3.8.3 1.3.8.4 1.3.8.5 1.3.8.6 1.3.8.71.3.8.8 1.3.8.9 1.3.9.1 1.3.9.2 1.3.9.3 1.3.9.4 1.3.9.5 1.3.9.6 1.3.9.71.3.9.8 1.3.9.9 1.4.1.1 1.4.1.2 1.4.1.3 1.4.1.4 1.4.1.5 1.4.1.6 1.4.1.71.4.1.8 1.4.1.9 1.4.2.1 1.4.2.2 1.4.2.3 1.4.2.4 1.4.2.5 1.4.2.6 1.4.2.71.4.2.8 1.4.2.9 1.4.3.1 1.4.3.2 1.4.3.3 1.4.3.4 1.4.3.5 1.4.3.6 1.4.3.71.4.3.8 1.4.3.9 1.4.4.1 1.4.4.2 1.4.4.3 1.4.4.4 1.4.4.5 1.4.4.6 1.4.4.71.4.4.8 1.4.4.9 1.4.5.1 1.4.5.2 1.4.5.3 1.4.5.4 1.4.5.5 1.4.5.6 1.4.5.71.4.5.8 1.4.5.9 1.4.6.1 1.4.6.2 1.4.6.3 1.4.6.4 1.4.6.5 1.4.6.6 1.4.6.71.4.6.8 1.4.6.9 1.4.7.1 1.4.7.2 1.4.7.3 1.4.7.4 1.4.7.5 1.4.7.6 1.4.7.71.4.7.8 1.4.7.9 1.4.8.1 1.4.8.2 1.4.8.3 1.4.8.4 1.4.8.5 1.4.8.6 1.4.8.71.4.8.8 1.4.8.9 1.4.9.1 1.4.9.2 1.4.9.3 1.4.9.4 1.4.9.5 1.4.9.6 1.4.9.71.4.9.8 1.4.9.9 1.5.1.1 1.5.1.2 1.5.1.3 1.5.1.4 1.5.1.5 1.5.1.6 1.5.1.71.5.1.8 1.5.1.9 1.5.2.1 1.5.2.2 1.5.2.3 1.5.2.4 1.5.2.5 1.5.2.6 1.5.2.71.5.2.8 1.5.2.9 1.5.3.1 1.5.3.2 1.5.3.3 1.5.3.4 1.5.3.5 1.5.3.6 1.5.3.71.5.3.8 1.5.3.9 1.5.4.1 1.5.4.2 1.5.4.3 1.5.4.4 1.5.4.5 1.5.4.6 1.5.4.71.5.4.8 1.5.4.9 1.5.5.1 1.5.5.2 1.5.5.3 1.5.5.4 1.5.5.5 1.5.5.6 1.5.5.71.5.5.8 1.5.5.9 1.5.6.1 1.5.6.2 1.5.6.3. 1.5.6.4 1.5.6.5 1.5.6.6 1.5.6.71.5.6.8 1.5.6.9 1.5.7.1 1.5.7.2 1.5.7.3 1.5.7.4 1.5.7.5 1.5.7.6 1.5.7.71.5.7.8 1.5.7.9 1.5.8.1 1.5.8.2 1.5.8.3 1.5.8.4 1.5.8.5 1.5.8.6 1.5.8.71.5.8.8 1.5.8.9 1.5.9.1 1.5.9.2 1.5.9.3 1.5.9.4 1.5.9.5 1.5.9.6 1.5.9.71.5.9.8 1.5.9.9 1.6.1.1 1.6.1.2 1.6.1.3 1.6.1.4 1.6.1.5 1.6.1.6 1.6.1.71.6.1.8 1.6.1.9 1.6.2.1 1.6.2.2 1.6.2.3 1.6.2.4 1.6.2.5 1.6.2.6 1.6.2.71.6.2.8 1.6.2.9 1.6.3.1 1.6.3.2 1.6.3.3 1.6.3.4 1.6.3.5 1.6.3.6 1.6.3.71.6.3.8 1.6.3.9 1.6.4.1 1.6.4.2 1.6.4.3 1.6.4.4 1.6.4.5 1.6.4.6 1.6.4.71.6.4.8 1.6.4.9 1.6.5.1 1.6.5.2 1.6.5.3 1.6.5.4 1.6.5.5 1.6.5.6 1.6.5.71.6.5.8 1.6.5.9 1.6.6.1 1.6.6.2 1.6.6.3 1.6.6.4 1.6.6.5 1.6.6.6 1.6.6.71.6.6.8 1.6.6.9 1.6.7.1 1.6.7.2 1.6.7.3 1.6.7.4 1.6.7.5 1.6.7.6 1.6.7.71.6.7.8 1.6.7.9 1.6.8.1 1.6.8.2 1.6.8.3 1.6.8.4. 1.6.8.5 1.6.8.6 1.6.8.71.6.8.8 1.6.8.9 1.6.9.1 1.6.9.2 1.6.9.3 1.6.9.4 1.6.9.5 1.6.9.6 1.6.9.71.6.9.8 1.6.9.9 1.7.1.1 1.7.1.2 1.7.1.3 1.7.1.4 1.7.1.5 1.7.1.6 1.7.1.71.7.1.8 1.7.1.9 1.7.2.1 1.7.2.2 1.7.2.3 1.7.2.4 1.7.2.5 1.7.2.6 1.7.2.71.7.2.8 1.7.2.9 1.7.3.1 1.7.3.2 1.7.3.3 1.7.3.4 1.7.3.5 1.7.3.6 1.7.3.71.7.3.8 1.7.3.9 1.7.4.1 1.7.4.2 1.7.4.3 1.7.4.4 1.7.4.5 1.7.4.6 1.7.4.71.7.4.8 1.7.4.9 1.7.5.1 1.7.5.2 1.7.5.3 1.7.5.4 1.7.5.5 1.7.5.6 1.7.5.71.7.5.8 1.7.5.9 1.7.6.1 1.7.6.2 1.7.6.3 1.7.6.4 1.7.6.5 1.7.6.6 1.7.6.71.7.6.8 1.7.6.9 1.7.7.1 1.7.7.2 1.7.7.3 1.7.7.4 1.7.7.5 1.7.7.6 1.7.7.71.7.7.8 1.7.7.9 1.7.8.1 1.7.8.2 1.7.8.3 1.7.8.4 1.7.8.5 1.7.8.6 1.7.8.71.7.8.8 1.7.8.9 1.7.9.1 1.7.9.2 1.7.9.3 1.7.9.4 1.7.9.5 1.7.9.6 1.7.9.71.7.9.8 1.7.9.9 1.8.1.1 1.8.1.2 1.8.1.3 1.8.1.4 1.8.1.5 1.8.1.6 1.8.1.71.8.1.8 1.8.1.9 1.8.2.1 1.8.2.2 1.8.2.3 1.8.2.4 1.8.2.5 1.8.2.6 1.8.2.71.8.2.8 1.8.2.9 1.8.3.1 1.8.3.2 1.8.3.3 1.8.3.4 1.8.3.5 1.8.3.6 1.8.3.71.8.3.8 1.8.3.9 1.8.4.1 1.8.4.2. 1.8.4.3. 1.8.4.4 1.8.4.5 1.8.4.61.8.4.7 1.8.4.8 1.8.4.9 1.8.5.1 1.8.5.2 1.8.5.3 1.8.5.4 1.8.5.5 1.8.5.61.8.5.7 1.8.5.8 1.8.5.9 1.8.6.1 1.8.6.2 1.8.6.3 1.8.6.4 1.8.6.5 1.8.6.61.8.6.7 1.8.6.8 1.8.6.9 1.8.7.1 1.8.7.2 1.8.7.3 1.8.7.4 1.8.7.5 1.8.7.61.8.7.7 1.8.7.8 1.8.7.9 1.8.8.1 1.8.8.2 1.8.8.3 1.8.8.4 1.8.8.5 1.8.8.61.8.8.7 1.8.8.8 1.8.8.9 1.8.9.1 1.8.9.2 1.8.9.3 1.8.9.4 1.8.9.5 1.8.9.61.8.9.7 1.8.9.8 1.8.9.9 1.9.1.1 1.9.1.2 1.9.1.3 1.9.1.4 1.9.1.5 1.9.1.61.9.1.7 1.9.1.8 1.9.1.9 1.9.2.1 1.9.2.2 1.9.2.3 1.9.2.4 1.9.2.5 1.9.2.61.9.2.7 1.9.2.8 1.9.2.9 1.9.3.1 1.9.3.2 1.9.3.3 1.9.3.4 1.9.3.5 1.9.3.61.9.3.7 1.9.3.8 1.9.3.9 1.9.4.1 1.9.4.2 1.9.4.3 1.9.4.4 1.9.4.5 1.9.4.61.9.4.7 1.9.4.8 1.9.4.9 1.9.5.1 1.9.5.2 1.9.5.3 1.9.5.4 1.9.5.5 1.9.5.61.9.5.7 1.9.5.8 1.9.5.9 1.9.6.1 1.9.6.2 1.9.6.3 1.9.6.4 1.9.6.5 1.9.6.61.9.6.7 1.9.6.8 1.9.6.9 1.9.7.1 1.9.7.2 1.9.7.3 1.9.7.4 1.9.7.5 1.9.7.61.9.7.7 1.9.7.8 1.9.7.9 1.9.8.1 1.9.8.2 1.9.8.3 1.9.8.4 1.9.8.5 1.9.8.61.9.8.7 1.9.8.8 1.9.8.9 1.9.9.1 1.9.9.2 1.9.9.3 1.9.9.4 1.9.9.5 1.9.9.61.9.9.7 1.9.9.8 1.9.9.9 2.1.1.1 2.1.1.2 2.1.1.3 2.1.1.4 2.1.1.5 2.1.1.62.1.1.7 2.1.1.8 2.1.1.9 2.1.2.1 2.1.2.2 2.1.2.3 2.1.2.4 2.1.2.5 2.1.2.62.1.2.7 2.1.2.8 2.1.2.9 2.1.3.1 2.1.3.2 2.1.3.3 2.1.3.4 2.1.3.5 2.1.3.62.1.3.7 2.1.3.8 2.1.3.9 2.1.4.1 2.1.4.2 2.1.4.3 2.1.4.4 2.1.4.5 2.1.4.62.1.4.7 2.1.4.8 2.1.4.9 2.1.5.1 2.1.5.2 2.1.5.3 2.1.5.4 2.1.5.5 2.1.5.62.1.5.7 2.1.5.8 2.1.5.9 2.1.6.1 2.1.6.2 2.1.6.3 2.1.6.4 2.1.6.5 2.1.6.62.1.6.7 2.1.6.8 2.1.6.9 2.1.7.1 2.1.7.2 2.1.7.3 2.1.7.4 2.1.7.5 2.1.7.62.1.7.7 2.1.7.8 2.1.7.9 2.1.8.1 2.1.8.2 2.1.8.3 2.1.8.4 2.1.8.5 2.1.8.62.1.8.7 2.1.8.8 2.1.8.9 2.1.9.1 2.1.9.2 2.1.9.3 2.1.9.4 2.1.9.5 2.1.9.62.1.9.7 2.1.9.8 2.1.9.9 2.2.1.1 2.2.1.2 2.2.1.3 2.2.1.4 2.2.1.5 2.2.1.62.2.1.7 2.2.1.8 2.2.1.9 2.2.2.1 2.2.2.2 2.2.2.3 2.2.2.4 2.2.2.5 2.2.2.62.2.2.7 2.2.2.8 2.2.2.9 2.2.3.1 2.2.3.2 2.2.3.3 2.2.3.4 2.2.3.5 2.2.3.62.2.3.7 2.2.3.8 2.2.3.9 2.2.4.1 2.2.4.2 2.2.4.3 2.2.4.4 2.2.4.5 2.2.4.62.2.4.7 2.2.4.8 2.2.4.9 2.2.5.1 2.2.5.2 2.2.5.3 2.2.5.4 2.2.5.5 2.2.5.62.2.5.7 2.2.5.8 2.2.5.9 2.2.6.1 2.2.6.2 2.2.6.3 2.2.6.4 2.2.6.5 2.2.6.62.2.6.7 2.2.6.8 2.2.6.9 2.2.7.1 2.2.7.2 2.2.7.3 2.2.7.4 2.2.7.5 2.2.7.62.2.7.7 2.2.7.8 2.2.7.9 2.2.8.1 2.2.8.2 2.2.8.3 2.2.8.4 2.2.8.5 2.2.8.62.2.8.7 2.2.8.8 2.2.8.9 2.2.9.1 2.2.9.2 2.2.9.3 2.2.9.4 2.2.9.5 2.2.9.62.2.9.7 2.2.9.8 2.2.9.9 2.3.1.1 2.3.1.2 2.3.1.3 2.3.1.4 2.3.1.5 2.3.1.62.3.1.7 2.3.1.8 2.3.1.9 2.3.2.1 2.3.2.2 2.3.2.3 2.3.2.4 2.3.2.5 2.3.2.62.3.2.7 2.3.2.8 2.3.2.9 2.3.3.1 2.3.3.2 2.3.3.3 2.3.3.4 2.3.3.5 2.3.3.62.3.3.7 2.3.3.8 2.3.3.9 2.3.4.1 2.3.4.2 2.3.4.3 2.3.4.4 2.3.4.5 2.3.4.62.3.4.7 2.3.4.8 2.3.4.9 2.3.5.1 2.3.5.2 2.3.5.3 2.3.5.4 2.3.5.5 2.3.5.62.3.5.7 2.3.5.8 2.3.5.9 2.3.6.1 2.3.6.2 2.3.6.3 2.3.6.4 2.3.6.5 2.3.6.62.3.6.7 2.3.6.8 2.3.6.9 2.3.7.1 2.3.7.2 2.3.7.3 2.3.7.4 2.3.7.5 2.3.7.62.3.7.7 2.3.7.8 2.3.7.9 2.3.8.1 2.3.8.2 2.3.8.3 2.3.8.4 2.3.8.5 2.3.8.62.3.8.7 2.3.8.8 2.3.8.9 2.3.9.1 2.3.9.2 2.3.9.3 2.3.9.4 2.3.9.5 2.3.9.62.3.9.7 2.3.9.8 2.3.9.9 2.4.1.1 2.4.1.2 2.4.1.3 2.4.1.4 2.4.1.5 2.4.1.62.4.1.7 2.4.1.8 2.4.1.9 2.4.2.1 2.4.2.2 2.4.2.3 2.4.2.4 2.4.2.5 2.4.2.62.4.2.7 2.4.2.8 2.4.2.9 2.4.3.1 2.4.3.2 2.4.3.3 2.4.3.4 2.4.3.5 2.4.3.62.4.3.7 2.4.3.8 2.4.3.9 2.4.4.1 2.4.4.2 2.4.4.3 2.4.4.4 2.4.4.5 2.4.4.62.4.4.7 2.4.4.8 2.4.4.9 2.4.5.1 2.4.5.2 2.4.5.3 2.4.5.4 2.4.5.5 2.4.5.62.4.5.7 2.4.5.8 2.4.5.9 2.4.6.1 2.4.6.2 2.4.6.3 2.4.6.4 2.4.6.5 2.4.6.62.4.6.7 2.4.6.8 2.4.6.9 2.4.7.1 2.4.7.2 2.4.7.3 2.4.7.4 2.4.7.5 2.4.7.62.4.7.7 2.4.7.8 2.4.7.9 2.4.8.1 2.4.8.2 2.4.8.3 2.4.8.4 2.4.8.5 2.4.8.62.4.8.7 2.4.8.8 2.4.8.9 2.4.9.1 2.4.9.2 2.4.9.3 2.4.9.4 2.4.9.5 2.4.9.62.4.9.7 2.4.9.8 2.4.9.9 2.5.1.1 2.5.1.2 2.5.1.3 2.5.1.4 2.5.1.5 2.5.1.62.5.1.7 2.5.1.8 2.5.1.9 2.5.2.1 2.5.2.2 2.5.2.3 2.5.2.4 2.5.2.5 2.5.2.62.5.2.7 2.5.2.8 2.5.2.9 2.5.3.1 2.5.3.2 2.5.3.3 2.5.3.4 2.5.3.5 2.5.3.62.5.3.7 2.5.3.8 2.5.3.9 2.5.4.1 2.5.4.2 2.5.4.3 2.5.4.4 2.5.4.5 2.5.4.62.5.4.7 2.5.4.8 2.5.4.9 2.5.5.1 2.5.5.2 2.5.5.3 2.5.5.4 2.5.5.5 2.5.5.62.5.5.7 2.5.5.8 2.5.5.9 2.5.6.1 2.5.6.2 2.5.6.3 2.5.6.4 2.5.6.5 2.5.6.62.5.6.7 2.5.6.8 2.5.6.9 2.5.7.1 2.5.7.2 2.5.7.3 2.5.7.4 2.5.7.5 2.5.7.62.5.7.7 2.5.7.8 2.5.7.9 2.5.8.1 2.5.8.2 2.5.8.3 2.5.8.4 2.5.8.5 2.5.8.62.5.8.7 2.5.8.8 2.5.8.9 2.5.9.1 2.5.9.2 2.5.9.3 2.5.9.4 2.5.9.5 2.5.9.62.5.9.7 2.5.9.8 2.5.9.9 2.6.1.1 2.6.1.2 2.6.1.3 2.6.1.4 2.6.1.5 2.6.1.62.6.1.7 2.6.1.8 2.6.1.9 2.6.2.1 2.6.2.2 2.6.2.3 2.6.2.4 2.6.2.5 2.6.2.62.6.2.7 2.6.2.8 2.6.2.9 2.6.3.1 2.6.3.2 2.6.3.3 2.6.3.4 2.6.3.5 2.6.3.62.6.3.7 2.6.3.8 2.6.3.9 2.6.4.1 2.6.4.2 2.6.4.3 2.6.4.4 2.6.4.5 2.6.4.62.6.4.7 2.6.4.8 2.6.4.9 2.6.5.1 2.6.5.2 2.6.5.3 2.6.5.4 2.6.5.5 2.6.5.62.6.5.7 2.6.5.8 2.6.5.9 2.6.6.1 2.6.6.2 2.6.6.3 2.6.6.4 2.6.6.5 2.6.6.62.6.6.7 2.6.6.8 2.6.6.9 2.6.7.1 2.6.7.2 2.6.7.3 2.6.7.4 2.6.7.5 2.6.7.62.6.7.7 2.6.7.8 2.6.7.9 2.6.8.1 2.6.8.2 2.6.8.3 2.6.8.4 2.6.8.5 2.6.8.62.6.8.7 2.6.8.8 2.6.8.9 2.6.9.1 2.6.9.2 2.6.9.3 2.6.9.4 2.6.9.5 2.6.9.62.6.9.7 2.6.9.8 2.6.9.9 2.7.1.1 2.7.1.2 2.7.1.3 2.7.1.4 2.7.1.5 2.7.1.62.7.1.7 2.7.1.8 2.7.1.9 2.7.2.1 2.7.2.2 2.7.2.3 2.7.2.4 2.7.2.5 2.7.2.62.7.2.7 2.7.2.8 2.7.2.9 2.7.3.1 2.7.3.2 2.7.3.3 2.7.3.4 2.7.3.5 2.7.3.62.7.3.7 2.7.3.8 2.7.3.9 2.7.4.1 2.7.4.2 2.7.4.3 2.7.4.4 2.7.4.5 2.7.4.62.7.4.7 2.7.4.8 2.7.4.9 2.7.5.1 2.7.5.2 2.7.5.3 2.7.5.4 2.7.5.5 2.7.5.62.7.5.7 2.7.5.8 2.7.5.9 2.7.6.1 2.7.6.2 2.7.6.3 2.7.6.4 2.7.6.5 2.7.6.62.7.6.7 2.7.6.8 2.7.6.9 2.7.7.1 2.7.7.2 2.7.7.3 2.7.7.4 2.7.7.5 2.7.7.62.7.7.7 2.7.7.8 2.7.7.9 2.7.8.1 2.7.8.2 2.7.8.3 2.7.8.4 2.7.8.5 2.7.8.62.7.8.7 2.7.8.8 2.7.8.9 2.7.9.1 2.7.9.2 2.7.9.3 2.7.9.4 2.7.9.5 2.7.9.62.7.9.7 2.7.9.8 2.7.9.9 2.8.1.1 2.8.1.2 2.8.1.3 2.8.1.4 2.8.1.5 2.8.1.62.8.1.7 2.8.1.8 2.8.1.9 2.8.2.1 2.8.2.2 2.8.2.3 2.8.2.4 2.8.2.5 2.8.2.62.8.2.7 2.8.2.8 2.8.2.9 2.8.3.1 2.8.3.2 2.8.3.3 2.8.3.4 2.8.3.5 2.8.3.62.8.3.7 2.8.3.8 2.8.3.9 2.8.4.1 2.8.4.2 2.8.4.3 2.8.4.4 2.8.4.5 2.8.4.62.8.4.7 2.8.4.8 2.8.4.9 2.8.5.1 2.8.5.2 2.8.5.3 2.8.5.4 2.8.5.5 2.8.5.62.8.5.7 2.8.5.8 2.8.5.9 2.8.6.1 2.8.6.2 2.8.6.3 2.8.6.4 2.8.6.5 2.8.6.62.8.6.7 2.8.6.8 2.8.6.9 2.8.7.1 2.8.7.2 2.8.7.3 2.8.7.4 2.8.7.5 2.8.7.62.8.7.7 2.8.7.8 2.8.7.9 2.8.8.1 2.8.8.2 2.8.8.3 2.8.8.4 2.8.8.5 2.8.8.62.8.8.7 2.8.8.8 2.8.8.9 2.8.9.1 2.8.9.2 2.8.9.3 2.8.9.4 2.8.9.5 2.8.9.62.8.9.7 2.8.9.8 2.8.9.9 2.9.1.1 2.9.1.2 2.9.1.3 2.9.1.4 2.9.1.5 2.9.1.62.9.1.7 2.9.1.8 2.9.1.9 2.9.2.1 2.9.2.2 2.9.2.3 2.9.2.4 2.9.2.5 2.9.2.62.9.2.7 2.9.2.8 2.9.2.9 2.9.3.1 2.9.3.2 2.9.3.3 2.9.3.4 2.9.3.5 2.9.3.62.9.3.7 2.9.3.8 2.9.3.9 2.9.4.1 2.9.4.2 2.9.4.3 2.9.4.4 2.9.4.5 2.9.4.62.9.4.7 2.9.4.8 2.9.4.9 2.9.5.1 2.9.5.2 2.9.5.3 2.9.5.4 2.9.5.5 2.9.5.62.9.5.7 2.9.5.8 2.9.5.9 2.9.6.1 2.9.6.2 2.9.6.3 2.9.6.4 2.9.6.5 2.9.6.62.9.6.7 2.9.6.8 2.9.6.9 2.9.7.1 2.9.7.2 2.9.7.3 2.9.7.4 2.9.7.5 2.9.7.62.9.7.7 2.9.7.8 2.9.7.9 2.9.8.1 2.9.8.2 2.9.8.3 2.9.8.4 2.9.8.5 2.9.8.62.9.8.7 2.9.8.8 2.9.8.9 2.9.9.1 2.9.9.2 2.9.9.3 2.9.9.4 2.9.9.5 2.9.9.62.9.9.7 2.9.9.8 2.9.9.9 3.1.1.1 3.1.1.2 3.1.1.3 3.1.1.4 3.1.1.5 3.1.1.63.1.1.7 3.1.1.8 3.1.1.9 3.1.2.1 3.1.2.2 3.1.2.3 3.1.2.4 3.1.2.5 3.1.2.63.1.2.7 3.1.2.8 3.1.2.9 3.1.3.1 3.1.3.2 3.1.3.3 3.1.3.4 3.1.3.5 3.1.3.63.1.3.7 3.1.3.8 3.1.3.9 3.1.4.1 3.1.4.2 3.1.4.3 3.1.4.4 3.1.4.5 3.1.4.63.1.4.7 3.1.4.8 3.1.4.9 3.1.5.1 3.1.5.2 3.1.5.3 3.1.5.4 3.1.5.5 3.1.5.63.1.5.7 3.1.5.8 3.1.5.9 3.1.6.1 3.1.6.2 3.1.6.3 3.1.6.4 3.1.6.5 3.1.6.63.1.6.7 3.1.6.8 3.1.6.9 3.1.7.1 3.1.7.2 3.1.7.3 3.1.7.4 3.1.7.5 3.1.7.63.1.7.7 3.1.7.8 3.1.7.9 3.1.8.1 3.1.8.2 3.1.8.3 3.1.8.4 3.1.8.5 3.1.8.63.1.8.7 3.1.8.8 3.1.8.9 3.1.9.1 3.1.9.2 3.1.9.3 3.1.9.4 3.1.9.5 3.1.9.63.1.9.7 3.1.9.8 3.1.9.9 3.2.1.1 3.2.1.2 3.2.1.3 3.2.1.4 3.2.1.5 3.2.1.63.2.1.7 3.2.1.8 3.2.1.9 3.2.2.1 3.2.2.2 3.2.2.3 3.2.2.4 3.2.2.5 3.2.2.63.2.2.7 3.2.2.8 3.2.2.9 3.2.3.1 3.2.3.2 3.2.3.3 3.2.3.4 3.2.3.5 3.2.3.63.2.3.7 3.2.3.8 3.2.3.9 3.2.4.1 3.2.4.2 3.2.4.3 3.2.4.4 3.2.4.5 3.2.4.63.2.4.7 3.2.4.8 3.2.4.9 3.2.5.1 3.2.5.2 3.2.5.3 3.2.5.4 3.2.5.5 3.2.5.63.2.5.7 3.2.5.8 3.2.5.9 3.2.6.1 3.2.6.2 3.2.6.3 3.2.6.4 3.2.6.5 3.2.6.63.2.6.7 3.2.6.8 3.2.6.9 3.2.7.1 3.2.7.2 3.2.7.3 3.2.7.4 3.2.7.5 3.2.7.63.2.7.7 3.2.7.8 3.2.7.9 3.2.8.1 3.2.8.2 3.2.8.3 3.2.8.4 3.2.8.5 3.2.8.63.2.8.7 3.2.8.8 3.2.8.9 3.2.9.1 3.2.9.2 3.2.9.3 3.2.9.4 3.2.9.5 3.2.9.63.2.9.7 3.2.9.8 3.2.9.9 3.3.1.1 3.3.1.2 3.3.1.3 3.3.1.4 3.3.1.5 3.3.1.63.3.1.7 3.3.1.8 3.3.1.9 3.3.2.1 3.3.2.2 3.3.2.3 3.3.2.4 3.3.2.5 3.3.2.63.3.2.7 3.3.2.8 3.3.2.9 3.3.3.1 3.3.3.2 3.3.3.3 3.3.3.4 3.3.3.5 3.3.3.63.3.3.7 3.3.3.8 3.3.3.9 3.3.4.1 3.3.4.2 3.3.4.3 3.3.4.4 3.3.4.5 3.3.4.63.3.4.7 3.3.4.8 3.3.4.9 3.3.5.1 3.3.5.2 3.3.5.3 3.3.5.4 3.3.5.5 3.3.5.63.3.5.7 3.3.5.8 3.3.5.9 3.3.6.1 3.3.6.2 3.3.6.3 3.3.6.4 3.3.6.5 3.3.6.63.3.6.7 3.3.6.8 3.3.6.9 3.3.7.1 3.3.7.2 3.3.7.3 3.3.7.4 3.3.7.5 3.3.7.63.3.7.7 3.3.7.8 3.3.7.9 3.3.8.1 3.3.8.2 3.3.8.3 3.3.8.4 3.3.8.5 3.3.8.63.3.8.7 3.3.8.8 3.3.8.9 3.3.9.1 3.3.9.2 3.3.9.3 3.3.9.4 3.3.9.5 3.3.9.63.3.9.7 3.3.9.8 3.3.9.9 3.4.1.1 3.4.1.2 3.4.1.3 3.4.1.4 3.4.1.5 3.4.1.63.4.1.7 3.4.1.8 3.4.1.9 3.4.2.1 3.4.2.2 3.4.2.3 3.4.2.4 3.4.2.5 3.4.2.63.4.2.7 3.4.2.8 3.4.2.9 3.4.3.1 3.4.3.2 3.4.3.3 3.4.3.4 3.4.3.5 3.4.3.63.4.3.7 3.4.3.8 3.4.3.9 3.4.4.1 3.4.4.2 3.4.4.3 3.4.4.4 3.4.4.5 3.4.4.63.4.4.7 3.4.4.8 3.4.4.9 3.4.5.1 3.4.5.2 3.4.5.3 3.4.5.4 3.4.5.5 3.4.5.63.4.5.7 3.4.5.8 3.4.5.9 3.4.6.1 3.4.6.2 3.4.6.3 3.4.6.4 3.4.6.5 3.4.6.63.4.6.7 3.4.6.8 3.4.6.9 3.4.7.1 3.4.7.2 3.4.7.3 3.4.7.4 3.4.7.5 3.4.7.63.4.7.7 3.4.7.8 3.4.7.9 3.4.8.1 3.4.8.2 3.4.8.3 3.4.8.4 3.4.8.5 3.4.8.63.4.8.7 3.4.8.8 3.4.8.9 3.4.9.1 3.4.9.2 3.4.9.3 3.4.9.4 3.4.9.5 3.4.9.63.4.9.7 3.4.9.8 3.4.9.9 3.5.1.1 3.5.1.2 3.5.1.3 3.5.1.4 3.5.1.5 3.5.1.63.5.1.7 3.5.1.8 3.5.1.9 3.5.2.1 3.5.2.2 3.5.2.3 3.5.2.4 3.5.2.5 3.5.2.63.5.2.7 3.5.2.8 3.5.2.9 3.5.3.1 3.5.3.2 3.5.3.3 3.5.3.4 3.5.3.5 3.5.3.63.5.3.7 3.5.3.8 3.5.3.9 3.5.4.1 3.5.4.2 3.5.4.3 3.5.4.4 3.5.4.5 3.5.4.63.5.4.7 3.5.4.8 3.5.4.9 3.5.5.1 3.5.5.2 3.5.5.3 3.5.5.4 3.5.5.5 3.5.5.63.5.5.7 3.5.5.8 3.5.5.9 3.5.6.1 3.5.6.2 3.5.6.3 3.5.6.4 3.5.6.5 3.5.6.63.5.6.7 3.5.6.8 3.5.6.9 3.5.7.1 3.5.7.2 3.5.7.3 3.5.7.4 3.5.7.5 3.5.7.63.5.7.7 3.5.7.8 3.5.7.9 3.5.8.1 3.5.8.2 3.5.8.3 3.5.8.4 3.5.8.5 3.5.8.63.5.8.7 3.5.8.8 3.5.8.9 3.5.9.1 3.5.9.2 3.5.9.3 3.5.9.4 3.5.9.5 3.5.9.63.5.9.7 3.5.9.8 3.5.9.9 3.6.1.1 3.6.1.2 3.6.1.3 3.6.1.4 3.6.1.5 3.6.1.63.6.1.7 3.6.1.8 3.6.1.9 3.6.2.1 3.6.2.2 3.6.2.3 3.6.2.4 3.6.2.5 3.6.2.63.6.2.7 3.6.2.8 3.6.2.9 3.6.3.1 3.6.3.2 3.6.3.3 3.6.3.4 3.6.3.5 3.6.3.63.6.3.7 3.6.3.8 3.6.3.9 3.6.4.1 3.6.4.2 3.6.4.3 3.6.4.4 3.6.4.5 3.6.4.63.6.4.7 3.6.4.8 3.6.4.9 3.6.5.1 3.6.5.2 3.6.5.3 3.6.5.4 3.6.5.5 3.6.5.63.6.5.7 3.6.5.8 3.6.5.9 3.6.6.1 3.6.6.2 3.6.6.3 3.6.6.4 3.6.6.5 3.6.6.63.6.6.7 3.6.6.8 3.6.6.9 3.6.7.1 3.6.7.2 3.6.7.3 3.6.7.4 3.6.7.5 3.6.7.63.6.7.7 3.6.7.8 3.6.7.9 3.6.8.1 3.6.8.2 3.6.8.3 3.6.8.4 3.6.8.5 3.6.8.63.6.8.7 3.6.8.8 3.6.8.9 3.6.9.1 3.6.9.2 3.6.9.3 3.6.9.4 3.6.9.5 3.6.9.63.6.9.7 3.6.9.8 3.6.9.9 3.7.1.1 3.7.1.2 3.7.1.3 3.7.1.4 3.7.1.5 3.7.1.63.7.1.7 3.7.1.8 3.7.1.9 3.7.2.1 3.7.2.2 3.7.2.3 3.7.2.4 3.7.2.5 3.7.2.63.7.2.7 3.7.2.8 3.7.2.9 3.7.3.1 3.7.3.2 3.7.3.3 3.7.3.4 3.7.3.5 3.7.3.63.7.3.7 3.7.3.8 3.7.3.9 3.7.4.1 3.7.4.2 3.7.4.3 3.7.4.4 3.7.4.5 3.7.4.63.7.4.7 3.7.4.8 3.7.4.9 3.7.5.1 3.7.5.2 3.7.5.3 3.7.5.4 3.7.5.5 3.7.5.63.7.5.7 3.7.5.8 3.7.5.9 3.7.6.1 3.7.6.2 3.7.6.3 3.7.6.4 3.7.6.5 3.7.6.63.7.6.7 3.7.6.8 3.7.6.9 3.7.7.1 3.7.7.2 3.7.7.3 3.7.7.4 3.7.7.5 3.7.7.63.7.7.7 3.7.7.8 3.7.7.9 3.7.8.1 3.7.8.2 3.7.8.3 3.7.8.4 3.7.8.5 3.7.8.63.7.8.7 3.7.8.8 3.7.8.9 3.7.9.1 3.7.9.2 3.7.9.3 3.7.9.4 3.7.9.5 3.7.9.63.7.9.7 3.7.9.8 3.7.9.9 3.8.1.1 3.8.1.2 3.8.1.3 3.8.1.4 3.8.1.5 3.8.1.63.8.1.7 3.8.1.8 3.8.1.9 3.8.2.1 3.8.2.2 3.8.2.3 3.8.2.4 3.8.2.5 3.8.2.63.8.2.7 3.8.2.8 3.8.2.9 3.8.3.1 3.8.3.2 3.8.3.3 3.8.3.4 3.8.3.5 3.8.3.63.8.3.7 3.8.3.8 3.8.3.9 3.8.4.1 3.8.4.2 3.8.4.3 3.8.4.4 3.8.4.5 3.8.4.63.8.4.7 3.8.4.8 3.8.4.9 3.8.5.1 3.8.5.2 3.8.5.3 3.8.5.4 3.8.5.5 3.8.5.63.8.5.7 3.8.5.8 3.8.5.9 3.8.6.1 3.8.6.2 3.8.6.3 3.8.6.4 3.8.6.5 3.8.6.63.8.6.7 3.8.6.8 3.8.6.9 3.8.7.1 3.8.7.2 3.8.7.3 3.8.7.4 3.8.7.5 3.8.7.63.8.7.7 3.8.7.8 3.8.7.9 3.8.8.1 3.8.8.2 3.8.8.3 3.8.8.4 3.8.8.5 3.8.8.63.8.8.7 3.8.8.8 3.8.8.9 3.8.9.1 3.8.9.2 3.8.9.3 3.8.9.4 3.8.9.5 3.8.9.63.8.9.7 3.8.9.8 3.8.9.9 3.9.1.1 3.9.1.2 3.9.1.3 3.9.1.4 3.9.1.5 3.9.1.63.9.1.7 3.9.1.8 3.9.1.9 3.9.2.1 3.9.2.2 3.9.2.3 3.9.2.4 3.9.2.5 3.9.2.63.9.2.7 3.9.2.8 3.9.2.9 3.9.3.1 3.9.3.2 3.9.3.3 3.9.3.4 3.9.3.5 3.9.3.63.9.3.7 3.9.3.8 3.9.3.9 3.9.4.1 3.9.4.2 3.9.4.3 3.9.4.4 3.9.4.5 3.9.4.63.9.4.7 3.9.4.8 3.9.4.9 3.9.5.1 3.9.5.2 3.9.5.3 3.9.5.4 3.9.5.5 3.9.5.63.9.5.7 3.9.5.8 3.9.5.9 3.9.6.1 3.9.6.2 3.9.6.3 3.9.6.4 3.9.6.5 3.9.6.63.9.6.7 3.9.6.8 3.9.6.9 3.9.7.1 3.9.7.2 3.9.7.3 3.9.7.4 3.9.7.5 3.9.7.63.9.7.7 3.9.7.8 3.9.7.9 3.9.8.1 3.9.8.2 3.9.8.3 3.9.8.4 3.9.8.5 3.9.8.63.9.8.7 3.9.8.8 3.9.8.9 3.9.9.1 3.9.9.2 3.9.9.3 3.9.9.4 3.9.9.5 3.9.9.63.9.9.7 3.9.9.8 3.9.9.9 4.1.1.1 4.1.1.2 4.1.1.3 4.1.1.4 4.1.1.5 4.1.1.64.1.1.7 4.1.1.8 4.1.1.9 4.1.2.1 4.1.2.2 4.1.2.3 4.1.2.4 4.1.2.5 4.1.2.64.1.2.7 4.1.2.8 4.1.2.9 4.1.3.1 4.1.3.2 4.1.3.3 4.1.3.4 4.1.3.5 4.1.3.64.1.3.7 4.1.3.8 4.1.3.9 4.1.4.1 4.1.4.2 4.1.4.3 4.1.4.4 4.1.4.5 4.1.4.64.1.4.7 4.1.4.8 4.1.4.9 4.1.5.1 4.1.5.2 4.1.5.3 4.1.5.4 4.1.5.5 4.1.5.64.1.5.7 4.1.5.8 4.1.5.9 4.1.6.1 4.1.6.2 4.1.6.3 4.1.6.4 4.1.6.5 4.1.6.64.1.6.7 4.1.6.8 4.1.6.9 4.1.7.1 4.1.7.2 4.1.7.3 4.1.7.4 4.1.7.5 4.1.7.64.1.7.7 4.1.7.8 4.1.7.9 4.1.8.1 4.1.8.2 4.1.8.3 4.1.8.4 4.1.8.5 4.1.8.64.1.8.7 4.1.8.8 4.1.8.9 4.1.9.1 4.1.9.2 4.1.9.3 4.1.9.4 4.1.9.5 4.1.9.64.1.9.7 4.1.9.8 4.1.9.9 4.2.1.1 4.2.1.2 4.2.1.3 4.2.1.4 4.2.1.5 4.2.1.64.2.1.7 4.2.1.8 4.2.1.9 4.2.2.1 4.2.2.2 4.2.2.3 4.2.2.4 4.2.2.5 4.2.2.64.2.2.7 4.2.2.8 4.2.2.9 4.2.3.1 4.2.3.2 4.2.3.3 4.2.3.4 4.2.3.5 4.2.3.64.2.3.7 4.2.3.8 4.2.3.9 4.2.4.1 4.2.4.2 4.2.4.3 4.2.4.4 4.2.4.5 4.2.4.64.2.4.7 4.2.4.8 4.2.4.9 4.2.5.1 4.2.5.2 4.2.5.3 4.2.5.4 4.2.5.5 4.2.5.64.2.5.7 4.2.5.8 4.2.5.9 4.2.6.1 4.2.6.2 4.2.6.3 4.2.6.4 4.2.6.5 4.2.6.64.2.6.7 4.2.6.8 4.2.6.9 4.2.7.1 4.2.7.2 4.2.7.3 4.2.7.4 4.2.7.5 4.2.7.64.2.7.7 4.2.7.8 4.2.7.9 4.2.8.1 4.2.8.2 4.2.8.3 4.2.8.4 4.2.8.5 4.2.8.64.2.8.7 4.2.8.8 4.2.8.9 4.2.9.1 4.2.9.2 4.2.9.3 4.2.9.4 4.2.9.5 4.2.9.64.2.9.7 4.2.9.8 4.2.9.9 4.3.1.1 4.3.1.2 4.3.1.3 4.3.1.4 4.3.1.5 4.3.1.64.3.1.7 4.3.1.8 4.3.1.9 4.3.2.1 4.3.2.2 4.3.2.3 4.3.2.4 4.3.2.5 4.3.2.64.3.2.7 4.3.2.8 4.3.2.9 4.3.3.1 4.3.3.2 4.3.3.3 4.3.3.4 4.3.3.5 4.3.3.64.3.3.7 4.3.3.8 4.3.3.9 4.3.4.1 4.3.4.2 4.3.4.3 4.3.4.4 4.3.4.5 4.3.4.64.3.4.7 4.3.4.8 4.3.4.9 4.3.5.1 4.3.5.2 4.3.5.3 4.3.5.4 4.3.5.5 4.3.5.64.3.5.7 4.3.5.8 4.3.5.9 4.3.6.1 4.3.6.2 4.3.6.3 4.3.6.4 4.3.6.5 4.3.6.64.3.6.7 4.3.6.8 4.3.6.9 4.3.7.1 4.3.7.2 4.3.7.3 4.3.7.4 4.3.7.5 4.3.7.64.3.7.7 4.3.7.8 4.3.7.9 4.3.8.1 4.3.8.2 4.3.8.3 4.3.8.4 4.3.8.5 4.3.8.64.3.8.7 4.3.8.8 4.3.8.9 4.3.9.1 4.3.9.2 4.3.9.3 4.3.9.4 4.3.9.5 4.3.9.64.3.9.7 4.3.9.8 4.3.9.9 4.4.1.1 4.4.1.2 4.4.1.3 4.4.1.4 4.4.1.5 4.4.1.64.4.1.7 4.4.1.8 4.4.1.9 4.4.2.1 4.4.2.2 4.4.2.3 4.4.2.4 4.4.2.5 4.4.2.64.4.2.7 4.4.2.8 4.4.2.9 4.4.3.1 4.4.3.2 4.4.3.3 4.4.3.4 4.4.3.5 4.4.3.64.4.3.7 4.4.3.8 4.4.3.9 4.4.4.1 4.4.4.2 4.4.4.3 4.4.4.4 4.4.4.5 4.4.4.64.4.4.7 4.4.4.8 4.4.4.9 4.4.5.1 4.4.5.2 4.4.5.3 4.4.5.4 4.4.5.5 4.4.5.64.4.5.7 4.4.5.8 4.4.5.9 4.4.6.1 4.4.6.2 4.4.6.3 4.4.6.4 4.4.6.5 4.4.6.64.4.6.7 4.4.6.8 4.4.6.9 4.4.7.1 4.4.7.2 4.4.7.3 4.4.7.4 4.4.7.5 4.4.7.64.4.7.7 4.4.7.8 4.4.7.9 4.4.8.1 4.4.8.2 4.4.8.3 4.4.8.4 4.4.8.5 4.4.8.64.4.8.7 4.4.8.8 4.4.8.9 4.4.9.1 4.4.9.2 4.4.9.3 4.4.9.4 4.4.9.5 4.4.9.64.4.9.7 4.4.9.8 4.4.9.9 4.5.1.1 4.5.1.2 4.5.1.3 4.5.1.4 4.5.1.5 4.5.1.64.5.1.7 4.5.1.8 4.5.1.9 4.5.2.1 4.5.2.2 4.5.2.3 4.5.2.4 4.5.2.5 4.5.2.64.5.2.7 4.5.2.8 4.5.2.9 4.5.3.1 4.5.3.2 4.5.3.3 4.5.3.4 4.5.3.5 4.5.3.64.5.3.7 4.5.3.8 4.5.3.9 4.5.4.1 4.5.4.2 4.5.4.3 4.5.4.4 4.5.4.5 4.5.4.64.5.4.7 4.5.4.8 4.5.4.9 4.5.5.1 4.5.5.2 4.5.5.3 4.5.5.4 4.5.5.5 4.5.5.64.5.5.7 4.5.5.8 4.5.5.9 4.5.6.1 4.5.6.2 4.5.6.3 4.5.6.4 4.5.6.5 4.5.6.64.5.6.7 4.5.6.8 4.5.6.9 4.5.7.1 4.5.7.2 4.5.7.3 4.5.7.4 4.5.7.5 4.5.7.64.5.7.7 4.5.7.8 4.5.7.9 4.5.8.1 4.5.8.2 4.5.8.3 4.5.8.4 4.5.8.5 4.5.8.64.5.8.7 4.5.8.8 4.5.8.9 4.5.9.1 4.5.9.2 4.5.9.3 4.5.9.4 4.5.9.5 4.5.9.64.5.9.7 4.5.9.8 4.5.9.9 4.6.1.1 4.6.1.2 4.6.1.3 4.6.1.4 4.6.1.5 4.6.1.64.6.1.7 4.6.1.8 4.6.1.9 4.6.2.1 4.6.2.2 4.6.2.3 4.6.2.4 4.6.2.5 4.6.2.64.6.2.7 4.6.2.8 4.6.2.9 4.6.3.1 4.6.3.2 4.6.3.3 4.6.3.4 4.6.3.5 4.6.3.64.6.3.7 4.6.3.8 4.6.3.9 4.6.4.1 4.6.4.2 4.6.4.3 4.6.4.4 4.6.4.5 4.6.4.64.6.4.7 4.6.4.8 4.6.4.9 4.6.5.1 4.6.5.2 4.6.5.3 4.6.5.4 4.6.5.5 4.6.5.64.6.5.7 4.6.5.8 4.6.5.9 4.6.6.1 4.6.6.2 4.6.6.3 4.6.6.4 4.6.6.5 4.6.6.64.6.6.7 4.6.6.8 4.6.6.9 4.6.7.1 4.6.7.2 4.6.7.3 4.6.7.4 4.6.7.5 4.6.7.64.6.7.7 4.6.7.8 4.6.7.9 4.6.8.1 4.6.8.2 4.6.8.3 4.6.8.4 4.6.8.5 4.6.8.64.6.8.7 4.6.8.8 4.6.8.9 4.6.9.1 4.6.9.2 4.6.9.3 4.6.9.4 4.6.9.5 4.6.9.64.6.9.7 4.6.9.8 4.6.9.9 4.7.1.1 4.7.1.2 4.7.1.3 4.7.1.4 4.7.1.5 4.7.1.64.7.1.7 4.7.1.8 4.7.1.9 4.7.2.1 4.7.2.2 4.7.2.3 4.7.2.4 4.7.2.5 4.7.2.64.7.2.7 4.7.2.8 4.7.2.9 4.7.3.1 4.7.3.2 4.7.3.3 4.7.3.4 4.7.3.5 4.7.3.64.7.3.7 4.7.3.8 4.7.3.9 4.7.4.1 4.7.4.2 4.7.4.3 4.7.4.4 4.7.4.5 4.7.4.64.7.4.7 4.7.4.8 4.7.4.9 4.7.5.1 4.7.5.2 4.7.5.3 4.7.5.4 4.7.5.5 4.7.5.64.7.5.7 4.7.5.8 4.7.5.9 4.7.6.1 4.7.6.2 4.7.6.3 4.7.6.4 4.7.6.5 4.7.6.64.7.6.7 4.7.6.8 4.7.6.9 4.7.7.1 4.7.7.2 4.7.7.3 4.7.7.4 4.7.7.5 4.7.7.64.7.7.7 4.7.7.8 4.7.7.9 4.7.8.1 4.7.8.2 4.7.8.3 4.7.8.4 4.7.8.5 4.7.8.64.7.8.7 4.7.8.8 4.7.8.9 4.7.9.1 4.7.9.2 4.7.9.3 4.7.9.4 4.7.9.5 4.7.9.64.7.9.7 4.7.9.8 4.7.9.9 4.8.1.1 4.8.1.2 4.8.1.3 4.8.1.4 4.8.1.5 4.8.1.64.8.1.7 4.8.1.8 4.8.1.9 4.8.2.1 4.8.2.2 4.8.2.3 4.8.2.4 4.8.2.5 4.8.2.64.8.2.7 4.8.2.8 4.8.2.9 4.8.3.1 4.8.3.2 4.8.3.3 4.8.3.4 4.8.3.5 4.8.3.64.8.3.7 4.8.3.8 4.8.3.9 4.8.4.1 4.8.4.2 4.8.4.3 4.8.4.4 4.8.4.5 4.8.4.64.8.4.7 4.8.4.8 4.8.4.9 4.8.5.1 4.8.5.2 4.8.5.3 4.8.5.4 4.8.5.5 4.8.5.64.8.5.7 4.8.5.8 4.8.5.9 4.8.6.1 4.8.6.2 4.8.6.3 4.8.6.4 4.8.6.5 4.8.6.64.8.6.7 4.8.6.8 4.8.6.9 4.8.7.1 4.8.7.2 4.8.7.3 4.8.7.4 4.8.7.5 4.8.7.64.8.7.7 4.8.7.8 4.8.7.9 4.8.8.1 4.8.8.2 4.8.8.3 4.8.8.4 4.8.8.5 4.8.8.64.8.8.7 4.8.8.8 4.8.8.9 4.8.9.1 4.8.9.2 4.8.9.3 4.8.9.4 4.8.9.5 4.8.9.64.8.9.7 4.8.9.8 4.8.9.9 4.9.1.1 4.9.1.2 4.9.1.3 4.9.1.4 4.9.1.5 4.9.1.64.9.1.7 4.9.1.8 4.9.1.9 4.9.2.1 4.9.2.2 4.9.2.3 4.9.2.4 4.9.2.5 4.9.2.64.9.2.7 4.9.2.8 4.9.2.9 4.9.3.1 4.9.3.2 4.9.3.3 4.9.3.4 4.9.3.5 4.9.3.64.9.3.7 4.9.3.8 4.9.3.9 4.9.4.1 4.9.4.2 4.9.4.3 4.9.4.4 4.9.4.5 4.9.4.64.9.4.7 4.9.4.8 4.9.4.9 4.9.5.1 4.9.5.2 4.9.5.3 4.9.5.4 4.9.5.5 4.9.5.64.9.5.7 4.9.5.8 4.9.5.9 4.9.6.1 4.9.6.2 4.9.6.3 4.9.6.4 4.9.6.5 4.9.6.64.9.6.7 4.9.6.8 4.9.6.9 4.9.7.1 4.9.7.2 4.9.7.3 4.9.7.4 4.9.7.5 4.9.7.64.9.7.7 4.9.7.8 4.9.7.9 4.9.8.1 4.9.8.2 4.9.8.3 4.9.8.4 4.9.8.5 4.9.8.64.9.8.7 4.9.8.8 4.9.8.9 4.9.9.1 4.9.9.2 4.9.9.3 4.9.9.4 4.9.9.5 4.9.9.64.9.9.7 4.9.9.8 4.9.9.9 5.1.1.1 5.1.1.2 5.1.1.3 5.1.1.4 5.1.1.5 5.1.1.65.1.1.7 5.1.1.8 5.1.1.9 5.1.2.1 5.1.2.2 5.1.2.3 5.1.2.4 5.1.2.5 5.1.2.65.1.2.7 5.1.2.8 5.1.2.9 5.1.3.1 5.1.3.2 5.1.3.3 5.1.3.4 5.1.3.5 5.1.3.65.1.3.7 5.1.3.8 5.1.3.9 5.1.4.1 5.1.4.2 5.1.4.3 5.1.4.4 5.1.4.5 5.1.4.65.1.4.7 5.1.4.8 5.1.4.9 5.1.5.1 5.1.5.2 5.1.5.3 5.1.5.4 5.1.5.5 5.1.5.65.1.5.7 5.1.5.8 5.1.5.9 5.1.6.1 5.1.6.2 5.1.6.3 5.1.6.4 5.1.6.5 5.1.6.65.1.6.7 5.1.6.8 5.1.6.9 5.1.7.1 5.1.7.2 5.1.7.3 5.1.7.4 5.1.7.5 5.1.7.65.1.7.7 5.1.7.8 5.1.7.9 5.1.8.1 5.1.8.2 5.1.8.3 5.1.8.4 5.1.8.5 5.1.8.65.1.8.7 5.1.8.8 5.1.8.9 5.1.9.1 5.1.9.2 5.1.9.3 5.1.9.4 5.1.9.5 5.1.9.65.1.9.7 5.1.9.8 5.1.9.9 5.2.1.1 5.2.1.2 5.2.1.3 5.2.1.4 5.2.1.5 5.2.1.65.2.1.7 5.2.1.8 5.2.1.9 5.2.2.1 5.2.2.2 5.2.2.3 5.2.2.4 5.2.2.5 5.2.2.65.2.2.7 5.2.2.8 5.2.2.9 5.2.3.1 5.2.3.2 5.2.3.3 5.2.3.4 5.2.3.5 5.2.3.65.2.3.7 5.2.3.8 5.2.3.9 5.2.4.1 5.2.4.2 5.2.4.3 5.2.4.4 5.2.4.5 5.2.4.65.2.4.7 5.2.4.8 5.2.4.9 5.2.5.1 5.2.5.2 5.2.5.3 5.2.5.4 5.2.5.5 5.2.5.65.2.5.7 5.2.5.8 5.2.5.9 5.2.6.1 5.2.6.2 5.2.6.3 5.2.6.4 5.2.6.5 5.2.6.65.2.6.7 5.2.6.8 5.2.6.9 5.2.7.1 5.2.7.2 5.2.7.3 5.2.7.4 5.2.7.5 5.2.7.65.2.7.7 5.2.7.8 5.2.7.9 5.2.8.1 5.2.8.2 5.2.8.3 5.2.8.4 5.2.8.5 5.2.8.65.2.8.7 5.2.8.8 5.2.8.9 5.2.9.1 5.2.9.2 5.2.9.3 5.2.9.4 5.2.9.5 5.2.9.65.2.9.7 5.2.9.8 5.2.9.9 5.3.1.1 5.3.1.2 5.3.1.3 5.3.1.4 5.3.1.5 5.3.1.65.3.1.7 5.3.1.8 5.3.1.9 5.3.2.1 5.3.2.2 5.3.2.3 5.3.2.4 5.3.2.5 5.3.2.65.3.2.7 5.3.2.8 5.3.2.9 5.3.3.1 5.3.3.2 5.3.3.3 5.3.3.4 5.3.3.5 5.3.3.65.3.3.7 5.3.3.8 5.3.3.9 5.3.4.1 5.3.4.2 5.3.4.3 5.3.4.4 5.3.4.5 5.3.4.65.3.4.7 5.3.4.8 5.3.4.9 5.3.5.1 5.3.5.2 5.3.5.3 5.3.5.4 5.3.5.5 5.3.5.65.3.5.7 5.3.5.8 5.3.5.9 5.3.6.1 5.3.6.2 5.3.6.3 5.3.6.4 5.3.6.5 5.3.6.65.3.6.7 5.3.6.8 5.3.6.9 5.3.7.1 5.3.7.2 5.3.7.3 5.3.7.4 5.3.7.5 5.3.7.65.3.7.7 5.3.7.8 5.3.7.9 5.3.8.1 5.3.8.2 5.3.8.3 5.3.8.4 5.3.8.5 5.3.8.65.3.8.7 5.3.8.8 5.3.8.9 5.3.9.1 5.3.9.2 5.3.9.3 5.3.9.4 5.3.9.5 5.3.9.65.3.9.7 5.3.9.8 5.3.9.9 5.4.1.1 5.4.1.2 5.4.1.3 5.4.1.4 5.4.1.5 5.4.1.65.4.1.7 5.4.1.8 5.4.1.9 5.4.2.1 5.4.2.2 5.4.2.3 5.4.2.4 5.4.2.5 5.4.2.65.4.2.7 5.4.2.8 5.4.2.9 5.4.3.1 5.4.3.2 5.4.3.3 5.4.3.4 5.4.3.5 5.4.3.65.4.3.7 5.4.3.8 5.4.3.9 5.4.4.1 5.4.4.2 5.4.4.3 5.4.4.4 5.4.4.5 5.4.4.65.4.4.7 5.4.4.8 5.4.4.9 5.4.5.1 5.4.5.2 5.4.5.3 5.4.5.4 5.4.5.5 5.4.5.65.4.5.7 5.4.5.8 5.4.5.9 5.4.6.1 5.4.6.2 5.4.6.3 5.4.6.4 5.4.6.5 5.4.6.65.4.6.7 5.4.6.8 5.4.6.9 5.4.7.1 5.4.7.2 5.4.7.3 5.4.7.4 5.4.7.5 5.4.7.65.4.7.7 5.4.7.8 5.4.7.9 5.4.8.1 5.4.8.2 5.4.8.3 5.4.8.4 5.4.8.5 5.4.8.65.4.8.7 5.4.8.8 5.4.8.9 5.4.9.1 5.4.9.2 5.4.9.3 5.4.9.4 5.4.9.5 5.4.9.65.4.9.7 5.4.9.8 5.4.9.9 5.5.1.1 5.5.1.2 5.5.1.3 5.5.1.4 5.5.1.5 5.5.1.65.5.1.7 5.5.1.8 5.5.1.9 5.5.2.1 5.5.2.2 5.5.2.3 5.5.2.4 5.5.2.5 5.5.2.65.5.2.7 5.5.2.8 5.5.2.9 5.5.3.1 5.5.3.2 5.5.3.3 5.5.3.4 5.5.3.5 5.5.3.65.5.3.7 5.5.3.8 5.5.3.9 5.5.4.1 5.5.4.2 5.5.4.3 5.5.4.4 5.5.4.5 5.5.4.65.5.4.7 5.5.4.8 5.5.4.9 5.5.5.1 5.5.5.2 5.5.5.3 5.5.5.4 5.5.5.5 5.5.5.65.5.5.7 5.5.5.8 5.5.5.9 5.5.6.1 5.5.6.2 5.5.6.3 5.5.6.4 5.5.6.5 5.5.6.65.5.6.7 5.5.6.8 5.5.6.9 5.5.7.1 5.5.7.2 5.5.7.3 5.5.7.4 5.5.7.5 5.5.7.65.5.7.7 5.5.7.8 5.5.7.9 5.5.8.1 5.5.8.2 5.5.8.3 5.5.8.4 5.5.8.5 5.5.8.65.5.8.7 5.5.8.8 5.5.8.9 5.5.9.1 5.5.9.2 5.5.9.3 5.5.9.4 5.5.9.5 5.5.9.65.5.9.7 5.5.9.8 5.5.9.9 5.6.1.1 5.6.1.2 5.6.1.3 5.6.1.4 5.6.1.5 5.6.1.65.6.1.7 5.6.1.8 5.6.1.9 5.6.2.1 5.6.2.2 5.6.2.3 5.6.2.4 5.6.2.5 5.6.2.65.6.2.7 5.6.2.8 5.6.2.9 5.6.3.1 5.6.3.2 5.6.3.3 5.6.3.4 5.6.3.5 5.6.3.65.6.3.7 5.6.3.8 5.6.3.9 5.6.4.1 5.6.4.2 5.6.4.3 5.6.4.4 5.6.4.5 5.6.4.65.6.4.7 5.6.4.8 5.6.4.9 5.6.5.1 5.6.5.2 5.6.5.3 5.6.5.4 5.6.5.5 5.6.5.65.6.5.7 5.6.5.8 5.6.5.9 5.6.6.1 5.6.6.2 5.6.6.3 5.6.6.4 5.6.6.5 5.6.6.65.6.6.7 5.6.6.8 5.6.6.9 5.6.7.1 5.6.7.2 5.6.7.3 5.6.7.4 5.6.7.5 5.6.7.65.6.7.7 5.6.7.8 5.6.7.9 5.6.8.1 5.6.8.2 5.6.8.3 5.6.8.4 5.6.8.5 5.6.8.65.6.8.7 5.6.8.8 5.6.8.9 5.6.9.1 5.6.9.2 5.6.9.3 5.6.9.4 5.6.9.5 5.6.9.65.6.9.7 5.6.9.8 5.6.9.9 5.7.1.1 5.7.1.2 5.7.1.3 5.7.1.4 5.7.1.5 5.7.1.65.7.1.7 5.7.1.8 5.7.1.9 5.7.2.1 5.7.2.2 5.7.2.3 5.7.2.4 5.7.2.5 5.7.2.65.7.2.7 5.7.2.8 5.7.2.9 5.7.3.1 5.7.3.2 5.7.3.3 5.7.3.4 5.7.3.5 5.7.3.65.7.3.7 5.7.3.8 5.7.3.9 5.7.4.1 5.7.4.2 5.7.4.3 5.7.4.4 5.7.4.5 5.7.4.65.7.4.7 5.7.4.8 5.7.4.9 5.7.5.1 5.7.5.2 5.7.5.3 5.7.5.4 5.7.5.5 5.7.5.65.7.5.7 5.7.5.8 5.7.5.9 5.7.6.1 5.7.6.2 5.7.6.3 5.7.6.4 5.7.6.5 5.7.6.65.7.6.7 5.7.6.8 5.7.6.9 5.7.7.1 5.7.7.2 5.7.7.3 5.7.7.4 5.7.7.5 5.7.7.65.7.7.7 5.7.7.8 5.7.7.9 5.7.8.1 5.7.8.2 5.7.8.3 5.7.8.4 5.7.8.5 5.7.8.65.7.8.7 5.7.8.8 5.7.8.9 5.7.9.1 5.7.9.2 5.7.9.3 5.7.9.4 5.7.9.5 5.7.9.65.7.9.7 5.7.9.8 5.7.9.9 5.8.1.1 5.8.1.2 5.8.1.3 5.8.1.4 5.8.1.5 5.8.1.65.8.1.7 5.8.1.8 5.8.1.9 5.8.2.1 5.8.2.2 5.8.2.3 5.8.2.4 5.8.2.5 5.8.2.65.8.2.7 5.8.2.8 5.8.2.9 5.8.3.1 5.8.3.2 5.8.3.3 5.8.3.4 5.8.3.5 5.8.3.65.8.3.7 5.8.3.8 5.8.3.9 5.8.4.1 5.8.4.2 5.8.4.3 5.8.4.4 5.8.4.5 5.8.4.65.8.4.7 5.8.4.8 5.8.4.9 5.8.5.1 5.8.5.2 5.8.5.3 5.8.5.4 5.8.5.5 5.8.5.65.8.5.7 5.8.5.8 5.8.5.9 5.8.6.1 5.8.6.2 5.8.6.3 5.8.6.4 5.8.6.5 5.8.6.65.8.6.7 5.8.6.8 5.8.6.9 5.8.7.1 5.8.7.2 5.8.7.3 5.8.7.4 5.8.7.5 5.8.7.65.8.7.7 5.8.7.8 5.8.7.9 5.8.8.1 5.8.8.2 5.8.8.3 5.8.8.4 5.8.8.5 5.8.8.65.8.8.7 5.8.8.8 5.8.8.9 5.8.9.1 5.8.9.2 5.8.9.3 5.8.9.4 5.8.9.5 5.8.9.65.8.9.7 5.8.9.8 5.8.9.9 5.9.1.1 5.9.1.2 5.9.1.3 5.9.1.4 5.9.1.5 5.9.1.65.9.1.7 5.9.1.8 5.9.1.9 5.9.2.1 5.9.2.2 5.9.2.3 5.9.2.4 5.9.2.5 5.9.2.65.9.2.7 5.9.2.8 5.9.2.9 5.9.3.1 5.9.3.2 5.9.3.3 5.9.3.4 5.9.3.5 5.9.3.65.9.3.7 5.9.3.8 5.9.3.9 5.9.4.1 5.9.4.2 5.9.4.3 5.9.4.4 5.9.4.5 5.9.4.65.9.4.7 5.9.4.8 5.9.4.9 5.9.5.1 5.9.5.2 5.9.5.3 5.9.5.4 5.9.5.5 5.9.5.65.9.5.7 5.9.5.8 5.9.5.9 5.9.6.1 5.9.6.2 5.9.6.3 5.9.6.4 5.9.6.5 5.9.6.65.9.6.7 5.9.6.8 5.9.6.9 5.9.7.1 5.9.7.2 5.9.7.3 5.9.7.4 5.9.7.5 5.9.7.65.9.7.7 5.9.7.8 5.9.7.9 5.9.8.1 5.9.8.2 5.9.8.3 5.9.8.4 5.9.8.5 5.9.8.65.9.8.7 5.9.8.8 5.9.8.9 5.9.9.1 5.9.9.2 5.9.9.3 5.9.9.4 5.9.9.5 5.9.9.65.9.9.7 5.9.9.8 5.9.9.9 6.1.1.1 6.1.1.2 6.1.1.3 6.1.1.4 6.1.1.5 6.1.1.66.1.1.7 6.1.1.8 6.1.1.9 6.1.2.1 6.1.2.2 6.1.2.3 6.1.2.4 6.1.2.5 6.1.2.66.1.2.7 6.1.2.8 6.1.2.9 6.1.3.1 6.1.3.2 6.1.3.3 6.1.3.4 6.1.3.5 6.1.3.66.1.3.7 6.1.3.8 6.1.3.9 6.1.4.1 6.1.4.2 6.1.4.3 6.1.4.4 6.1.4.5 6.1.4.66.1.4.7 6.1.4.8 6.1.4.9 6.1.5.1 6.1.5.2 6.1.5.3 6.1.5.4 6.1.5.5 6.1.5.66.1.5.7 6.1.5.8 6.1.5.9 6.1.6.1 6.1.6.2 6.1.6.3 6.1.6.4 6.1.6.5 6.1.6.66.1.6.7 6.1.6.8 6.1.6.9 6.1.7.1 6.1.7.2 6.1.7.3 6.1.7.4 6.1.7.5 6.1.7.66.1.7.7 6.1.7.8 6.1.7.9 6.1.8.1 6.1.8.2 6.1.8.3 6.1.8.4 6.1.8.5 6.1.8.66.1.8.7 6.1.8.8 6.1.8.9 6.1.9.1 6.1.9.2 6.1.9.3 6.1.9.4 6.1.9.5 6.1.9.66.1.9.7 6.1.9.8 6.1.9.9 6.2.1.1 6.2.1.2 6.2.1.3 6.2.1.4 6.2.1.5 6.2.1.66.2.1.7 6.2.1.8 6.2.1.9 6.2.2.1 6.2.2.2 6.2.2.3 6.2.2.4 6.2.2.5 6.2.2.66.2.2.7 6.2.2.8 6.2.2.9 6.2.3.1 6.2.3.2 6.2.3.3 6.2.3.4 6.2.3.5 6.2.3.66.2.3.7 6.2.3.8 6.2.3.9 6.2.4.1 6.2.4.2 6.2.4.3 6.2.4.4 6.2.4.5 6.2.4.66.2.4.7 6.2.4.8 6.2.4.9 6.2.5.1 6.2.5.2 6.2.5.3 6.2.5.4 6.2.5.5 6.2.5.66.2.5.7 6.2.5.8 6.2.5.9 6.2.6.1 6.2.6.2 6.2.6.3 6.2.6.4 6.2.6.5 6.2.6.66.2.6.7 6.2.6.8 6.2.6.9 6.2.7.1 6.2.7.2 6.2.7.3 6.2.7.4 6.2.7.5 6.2.7.66.2.7.7 6.2.7.8 6.2.7.9 6.2.8.1 6.2.8.2 6.2.8.3 6.2.8.4 6.2.8.5 6.2.8.66.2.8.7 6.2.8.8 6.2.8.9 6.2.9.1 6.2.9.2 6.2.9.3 6.2.9.4 6.2.9.5 6.2.9.66.2.9.7 6.2.9.8 6.2.9.9 6.3.1.1 6.3.1.2 6.3.1.3 6.3.1.4 6.3.1.5 6.3.1.66.3.1.7 6.3.1.8 6.3.1.9 6.3.2.1 6.3.2.2 6.3.2.3 6.3.2.4 6.3.2.5 6.3.2.66.3.2.7 6.3.2.8 6.3.2.9 6.3.3.1 6.3.3.2 6.3.3.3 6.3.3.4 6.3.3.5 6.3.3.66.3.3.7 6.3.3.8 6.3.3.9 6.3.4.1 6.3.4.2 6.3.4.3 6.3.4.4 6.3.4.5 6.3.4.66.3.4.7 6.3.4.8 6.3.4.9 6.3.5.1 6.3.5.2 6.3.5.3 6.3.5.4 6.3.5.5 6.3.5.66.3.5.7 6.3.5.8 6.3.5.9 6.3.6.1 6.3.6.2 6.3.6.3 6.3.6.4 6.3.6.5 6.3.6.66.3.6.7 6.3.6.8 6.3.6.9 6.3.7.1 6.3.7.2 6.3.7.3 6.3.7.4 6.3.7.5 6.3.7.66.3.7.7 6.3.7.8 6.3.7.9 6.3.8.1 6.3.8.2 6.3.8.3 6.3.8.4 6.3.8.5 6.3.8.66.3.8.7 6.3.8.8 6.3.8.9 6.3.9.1 6.3.9.2 6.3.9.3 6.3.9.4 6.3.9.5 6.3.9.66.3.9.7 6.3.9.8 6.3.9.9 6.4.1.1 6.4.1.2 6.4.1.3 6.4.1.4 6.4.1.5 6.4.1.66.4.1.7 6.4.1.8 6.4.1.9 6.4.2.1 6.4.2.2 6.4.2.3 6.4.2.4 6.4.2.5 6.4.2.66.4.2.7 6.4.2.8 6.4.2.9 6.4.3.1 6.4.3.2 6.4.3.3 6.4.3.4 6.4.3.5 6.4.3.66.4.3.7 6.4.3.8 6.4.3.9 6.4.4.1 6.4.4.2 6.4.4.3 6.4.4.4 6.4.4.5 6.4.4.66.4.4.7 6.4.4.8 6.4.4.9 6.4.5.1 6.4.5.2 6.4.5.3 6.4.5.4 6.4.5.5 6.4.5.66.4.5.7 6.4.5.8 6.4.5.9 6.4.6.1 6.4.6.2 6.4.6.3 6.4.6.4 6.4.6.5 6.4.6.66.4.6.7 6.4.6.8 6.4.6.9 6.4.7.1 6.4.7.2 6.4.7.3 6.4.7.4 6.4.7.5 6.4.7.66.4.7.7 6.4.7.8 6.4.7.9 6.4.8.1 6.4.8.2 6.4.8.3 6.4.8.4 6.4.8.5 6.4.8.66.4.8.7 6.4.8.8 6.4.8.9 6.4.9.1 6.4.9.2 6.4.9.3 6.4.9.4 6.4.9.5 6.4.9.66.4.9.7 6.4.9.8 6.4.9.9 6.5.1.1 6.5.1.2 6.5.1.3 6.5.1.4 6.5.1.5 6.5.1.66.5.1.7 6.5.1.8 6.5.1.9 6.5.2.1 6.5.2.2 6.5.2.3 6.5.2.4 6.5.2.5 6.5.2.66.5.2.7 6.5.2.8 6.5.2.9 6.5.3.1 6.5.3.2 6.5.3.3 6.5.3.4 6.5.3.5 6.5.3.66.5.3.7 6.5.3.8 6.5.3.9 6.5.4.1 6.5.4.2 6.5.4.3 6.5.4.4 6.5.4.5 6.5.4.66.5.4.7 6.5.4.8 6.5.4.9 6.5.5.1 6.5.5.2 6.5.5.3 6.5.5.4 6.5.5.5 6.5.5.66.5.5.7 6.5.5.8 6.5.5.9 6.5.6.1 6.5.6.2 6.5.6.3 6.5.6.4 6.5.6.5 6.5.6.66.5.6.7 6.5.6.8 6.5.6.9 6.5.7.1 6.5.7.2 6.5.7.3 6.5.7.4 6.5.7.5 6.5.7.66.5.7.7 6.5.7.8 6.5.7.9 6.5.8.1 6.5.8.2 6.5.8.3 6.5.8.4 6.5.8.5 6.5.8.66.5.8.7 6.5.8.8 6.5.8.9 6.5.9.1 6.5.9.2 6.5.9.3 6.5.9.4 6.5.9.5 6.5.9.66.5.9.7 6.5.9.8 6.5.9.9 6.6.1.1 6.6.1.2 6.6.1.3 6.6.1.4 6.6.1.5 6.6.1.66.6.1.7 6.6.1.8 6.6.1.9 6.6.2.1 6.6.2.2 6.6.2.3 6.6.2.4 6.6.2.5 6.6.2.66.6.2.7 6.6.2.8 6.6.2.9 6.6.3.1 6.6.3.2 6.6.3.3 6.6.3.4 6.6.3.5 6.6.3.66.6.3.7 6.6.3.8 6.6.3.9 6.6.4.1 6.6.4.2 6.6.4.3 6.6.4.4 6.6.4.5 6.6.4.66.6.4.7 6.6.4.8 6.6.4.9 6.6.5.1 6.6.5.2 6.6.5.3 6.6.5.4 6.6.5.5 6.6.5.66.6.5.7 6.6.5.8 6.6.5.9 6.6.6.1 6.6.6.2 6.6.6.3 6.6.6.4 6.6.6.5 6.6.6.66.6.6.7 6.6.6.8 6.6.6.9 6.6.7.1 6.6.7.2 6.6.7.3 6.6.7.4 6.6.7.5 6.6.7.66.6.7.7 6.6.7.8 6.6.7.9 6.6.8.1 6.6.8.2 6.6.8.3 6.6.8.4 6.6.8.5 6.6.8.66.6.8.7 6.6.8.8 6.6.8.9 6.6.9.1 6.6.9.2 6.6.9.3 6.6.9.4 6.6.9.5 6.6.9.66.6.9.7 6.6.9.8 6.6.9.9 6.7.1.1 6.7.1.2 6.7.1.3 6.7.1.4 6.7.1.5 6.7.1.66.7.1.7 6.7.1.8 6.7.1.9 6.7.2.1 6.7.2.2 6.7.2.3 6.7.2.4 6.7.2.5 6.7.2.66.7.2.7 6.7.2.8 6.7.2.9 6.7.3.1 6.7.3.2 6.7.3.3 6.7.3.4 6.7.3.5 6.7.3.66.7.3.7 6.7.3.8 6.7.3.9 6.7.4.1 6.7.4.2 6.7.4.3 6.7.4.4 6.7.4.5 6.7.4.66.7.4.7 6.7.4.8 6.7.4.9 6.7.5.1 6.7.5.2 6.7.5.3 6.7.5.4 6.7.5.5 6.7.5.66.7.5.7 6.7.5.8 6.7.5.9 6.7.6.1 6.7.6.2 6.7.6.3 6.7.6.4 6.7.6.5 6.7.6.66.7.6.7 6.7.6.8 6.7.6.9 6.7.7.1 6.7.7.2 6.7.7.3 6.7.7.4 6.7.7.5 6.7.7.66.7.7.7 6.7.7.8 6.7.7.9 6.7.8.1 6.7.8.2 6.7.8.3 6.7.8.4 6.7.8.5 6.7.8.66.7.8.7 6.7.8.8 6.7.8.9 6.7.9.1 6.7.9.2 6.7.9.3 6.7.9.4 6.7.9.5 6.7.9.66.7.9.7 6.7.9.8 6.7.9.9 6.8.1.1 6.8.1.2 6.8.1.3 6.8.1.4 6.8.1.5 6.8.1.66.8.1.7 6.8.1.8 6.8.1.9 6.8.2.1 6.8.2.2 6.8.2.3 6.8.2.4 6.8.2.5 6.8.2.66.8.2.7 6.8.2.8 6.8.2.9 6.8.3.1 6.8.3.2 6.8.3.3 6.8.3.4 6.8.3.5 6.8.3.66.8.3.7 6.8.3.8 6.8.3.9 6.8.4.1 6.8.4.2 6.8.4.3 6.8.4.4 6.8.4.5 6.8.4.66.8.4.7 6.8.4.8 6.8.4.9 6.8.5.1 6.8.5.2 6.8.5.3 6.8.5.4 6.8.5.5 6.8.5.66.8.5.7 6.8.5.8 6.8.5.9 6.8.6.1 6.8.6.2 6.8.6.3 6.8.6.4 6.8.6.5 6.8.6.66.8.6.7 6.8.6.8 6.8.6.9 6.8.7.1 6.8.7.2 6.8.7.3 6.8.7.4 6.8.7.5 6.8.7.66.8.7.7 6.8.7.8 6.8.7.9 6.8.8.1 6.8.8.2 6.8.8.3 6.8.8.4 6.8.8.5 6.8.8.66.8.8.7 6.8.8.8 6.8.8.9 6.8.9.1 6.8.9.2 6.8.9.3 6.8.9.4 6.8.9.5 6.8.9.66.8.9.7 6.8.9.8 6.8.9.9 6.9.1.1 6.9.1.2 6.9.1.3 6.9.1.4 6.9.1.5 6.9.1.66.9.1.7 6.9.1.8 6.9.1.9 6.9.2.1 6.9.2.2 6.9.2.3 6.9.2.4 6.9.2.5 6.9.2.66.9.2.7 6.9.2.8 6.9.2.9 6.9.3.1 6.9.3.2 6.9.3.3 6.9.3.4 6.9.3.5 6.9.3.66.9.3.7 6.9.3.8 6.9.3.9 6.9.4.1 6.9.4.2 6.9.4.3 6.9.4.4 6.9.4.5 6.9.4.66.9.4.7 6.9.4.8 6.9.4.9 6.9.5.1 6.9.5.2 6.9.5.3 6.9.5.4 6.9.5.5 6.9.5.66.9.5.7 6.9.5.8 6.9.5.9 6.9.6.1 6.9.6.2 6.9.6.3 6.9.6.4 6.9.6.5 6.9.6.66.9.6.7 6.9.6.8 6.9.6.9 6.9.7.1 6.9.7.2 6.9.7.3 6.9.7.4 6.9.7.5 6.9.7.66.9.7.7 6.9.7.8 6.9.7.9 6.9.8.1 6.9.8.2 6.9.8.3 6.9.8.4 6.9.8.5 6.9.8.66.9.8.7 6.9.8.8 6.9.8.9 6.9.9.1 6.9.9.2 6.9.9.3 6.9.9.4 6.9.9.5 6.9.9.66.9.9.7 6.9.9.8 6.9.9.9 7.1.1.1 7.1.1.2 7.1.1.3 7.1.1.4 7.1.1.5 7.1.1.67.1.1.7 7.1.1.8 7.1.1.9 7.1.2.1 7.1.2.2 7.1.2.3 7.1.2.4 7.1.2.5 7.1.2.67.1.2.7 7.1.2.8 7.1.2.9 7.1.3.1 7.1.3.2 7.1.3.3 7.1.3.4 7.1.3.5 7.1.3.67.1.3.7 7.1.3.8 7.1.3.9 7.1.4.1 7.1.4.2 7.1.4.3 7.1.4.4 7.1.4.5 7.1.4.67.1.4.7 7.1.4.8 7.1.4.9 7.1.5.1 7.1.5.2 7.1.5.3 7.1.5.4 7.1.5.5 7.1.5.67.1.5.7 7.1.5.8 7.1.5.9 7.1.6.1 7.1.6.2 7.1.6.3 7.1.6.4 7.1.6.5 7.1.6.67.1.6.7 7.1.6.8 7.1.6.9 7.1.7.1 7.1.7.2 7.1.7.3 7.1.7.4 7.1.7.5 7.1.7.67.1.7.7 7.1.7.8 7.1.7.9 7.1.8.1 7.1.8.2 7.1.8.3 7.1.8.4 7.1.8.5 7.1.8.67.1.8.7 7.1.8.8 7.1.8.9 7.1.9.1 7.1.9.2 7.1.9.3 7.1.9.4 7.1.9.5 7.1.9.67.1.9.7 7.1.9.8 7.1.9.9 7.2.1.1 7.2.1.2 7.2.1.3 7.2.1.4 7.2.1.5 7.2.1.67.2.1.7 7.2.1.8 7.2.1.9 7.2.2.1 7.2.2.2 7.2.2.3 7.2.2.4 7.2.2.5 7.2.2.67.2.2.7 7.2.2.8 7.2.2.9 7.2.3.1 7.2.3.2 7.2.3.3 7.2.3.4 7.2.3.5 7.2.3.67.2.3.7 7.2.3.8 7.2.3.9 7.2.4.1 7.2.4.2 7.2.4.3 7.2.4.4 7.2.4.5 7.2.4.67.2.4.7 7.2.4.8 7.2.4.9 7.2.5.1 7.2.5.2 7.2.5.3 7.2.5.4 7.2.5.5 7.2.5.67.2.5.7 7.2.5.8 7.2.5.9 7.2.6.1 7.2.6.2 7.2.6.3 7.2.6.4 7.2.6.5 7.2.6.67.2.6.7 7.2.6.8 7.2.6.9 7.2.7.1 7.2.7.2 7.2.7.3 7.2.7.4 7.2.7.5 7.2.7.67.2.7.7 7.2.7.8 7.2.7.9 7.2.8.1 7.2.8.2 7.2.8.3 7.2.8.4 7.2.8.5 7.2.8.67.2.8.7 7.2.8.8 7.2.8.9 7.2.9.1 7.2.9.2 7.2.9.3 7.2.9.4 7.2.9.5 7.2.9.67.2.9.7 7.2.9.8 7.2.9.9 7.3.1.1 7.3.1.2 7.3.1.3 7.3.1.4 7.3.1.5 7.3.1.67.3.1.7 7.3.1.8 7.3.1.9 7.3.2.1 7.3.2.2 7.3.2.3 7.3.2.4 7.3.2.5 7.3.2.67.3.2.7 7.3.2.8 7.3.2.9 7.3.3.1 7.3.3.2 7.3.3.3 7.3.3.4 7.3.3.5 7.3.3.67.3.3.7 7.3.3.8 7.3.3.9 7.3.4.1 7.3.4.2 7.3.4.3 7.3.4.4 7.3.4.5 7.3.4.67.3.4.7 7.3.4.8 7.3.4.9 7.3.5.1 7.3.5.2 7.3.5.3 7.3.5.4 7.3.5.5 7.3.5.67.3.5.7 7.3.5.8 7.3.5.9 7.3.6.1 7.3.6.2 7.3.6.3 7.3.6.4 7.3.6.5 7.3.6.67.3.6.7 7.3.6.8 7.3.6.9 7.3.7.1 7.3.7.2 7.3.7.3 7.3.7.4 7.3.7.5 7.3.7.67.3.7.7 7.3.7.8 7.3.7.9 7.3.8.1 7.3.8.2 7.3.8.3 7.3.8.4 7.3.8.5 7.3.8.67.3.8.7 7.3.8.8 7.3.8.9 7.3.9.1 7.3.9.2 7.3.9.3 7.3.9.4 7.3.9.5 7.3.9.67.3.9.7 7.3.9.8 7.3.9.9 7.4.1.1 7.4.1.2 7.4.1.3 7.4.1.4 7.4.1.5 7.4.1.67.4.1.7 7.4.1.8 7.4.1.9 7.4.2.1 7.4.2.2 7.4.2.3 7.4.2.4 7.4.2.5 7.4.2.67.4.2.7 7.4.2.8 7.4.2.9 7.4.3.1 7.4.3.2 7.4.3.3 7.4.3.4 7.4.3.5 7.4.3.67.4.3.7 7.4.3.8 7.4.3.9 7.4.4.1 7.4.4.2 7.4.4.3 7.4.4.4 7.4.4.5 7.4.4.67.4.4.7 7.4.4.8 7.4.4.9 7.4.5.1 7.4.5.2 7.4.5.3 7.4.5.4 7.4.5.5 7.4.5.67.4.5.7 7.4.5.8 7.4.5.9 7.4.6.1 7.4.6.2 7.4.6.3 7.4.6.4 7.4.6.5 7.4.6.67.4.6.7 7.4.6.8 7.4.6.9 7.4.7.1 7.4.7.2 7.4.7.3 7.4.7.4 7.4.7.5 7.4.7.67.4.7.7 7.4.7.8 7.4.7.9 7.4.8.1 7.4.8.2 7.4.8.3 7.4.8.4 7.4.8.5 7.4.8.67.4.8.7 7.4.8.8 7.4.8.9 7.4.9.1 7.4.9.2 7.4.9.3 7.4.9.4 7.4.9.5 7.4.9.67.4.9.7 7.4.9.8 7.4.9.9 7.5.1.1 7.5.1.2 7.5.1.3 7.5.1.4 7.5.1.5 7.5.1.67.5.1.7 7.5.1.8 7.5.1.9 7.5.2.1 7.5.2.2 7.5.2.3 7.5.2.4 7.5.2.5 7.5.2.67.5.2.7 7.5.2.8 7.5.2.9 7.5.3.1 7.5.3.2 7.5.3.3 7.5.3.4 7.5.3.5 7.5.3.67.5.3.7 7.5.3.8 7.5.3.9 7.5.4.1 7.5.4.2 7.5.4.3 7.5.4.4 7.5.4.5 7.5.4.67.5.4.7 7.5.4.8 7.5.4.9 7.5.5.1 7.5.5.2 7.5.5.3 7.5.5.4 7.5.5.5 7.5.5.67.5.5.7 7.5.5.8 7.5.5.9 7.5.6.1 7.5.6.2 7.5.6.3 7.5.6.4 7.5.6.5 7.5.6.67.5.6.7 7.5.6.8 7.5.6.9 7.5.7.1 7.5.7.2 7.5.7.3 7.5.7.4 7.5.7.5 7.5.7.67.5.7.7 7.5.7.8 7.5.7.9 7.5.8.1 7.5.8.2 7.5.8.3 7.5.8.4 7.5.8.5 7.5.8.67.5.8.7 7.5.8.8 7.5.8.9 7.5.9.1 7.5.9.2 7.5.9.3 7.5.9.4 7.5.9.5 7.5.9.67.5.9.7 7.5.9.8 7.5.9.9 7.6.1.1 7.6.1.2 7.6.1.3 7.6.1.4 7.6.1.5 7.6.1.67.6.1.7 7.6.1.8 7.6.1.9 7.6.2.1 7.6.2.2 7.6.2.3 7.6.2.4 7.6.2.5 7.6.2.67.6.2.7 7.6.2.8 7.6.2.9 7.6.3.1 7.6.3.2 7.6.3.3 7.6.3.4 7.6.3.5 7.6.3.67.6.3.7 7.6.3.8 7.6.3.9 7.6.4.1 7.6.4.2 7.6.4.3 7.6.4.4 7.6.4.5 7.6.4.67.6.4.7 7.6.4.8 7.6.4.9 7.6.5.1 7.6.5.2 7.6.5.3 7.6.5.4 7.6.5.5 7.6.5.67.6.5.7 7.6.5.8 7.6.5.9 7.6.6.1 7.6.6.2 7.6.6.3 7.6.6.4 7.6.6.5 7.6.6.67.6.6.7 7.6.6.8 7.6.6.9 7.6.7.1 7.6.7.2 7.6.7.3 7.6.7.4 7.6.7.5 7.6.7.67.6.7.7 7.6.7.8 7.6.7.9 7.6.8.1 7.6.8.2 7.6.8.3 7.6.8.4 7.6.8.5 7.6.8.67.6.8.7 7.6.8.8 7.6.8.9 7.6.9.1 7.6.9.2 7.6.9.3 7.6.9.4 7.6.9.5 7.6.9.67.6.9.7 7.6.9.8 7.6.9.9 7.7.1.1 7.7.1.2 7.7.1.3 7.7.1.4 7.7.1.5 7.7.1.67.7.1.7 7.7.1.8 7.7.1.9 7.7.2.1 7.7.2.2 7.7.2.3 7.7.2.4 7.7.2.5 7.7.2.67.7.2.7 7.7.2.8 7.7.2.9 7.7.3.1 7.7.3.2 7.7.3.3 7.7.3.4 7.7.3.5 7.7.3.67.7.3.7 7.7.3.8 7.7.3.9 7.7.4.1 7.7.4.2 7.7.4.3 7.7.4.4 7.7.4.5 7.7.4.67.7.4.7 7.7.4.8 7.7.4.9 7.7.5.1 7.7.5.2 7.7.5.3 7.7.5.4 7.7.5.5 7.7.5.67.7.5.7 7.7.5.8 7.7.5.9 7.7.6.1 7.7.6.2 7.7.6.3 7.7.6.4 7.7.6.5 7.7.6.67.7.6.7 7.7.6.8 7.7.6.9 7.7.7.1 7.7.7.2 7.7.7.3 7.7.7.4 7.7.7.5 7.7.7.67.7.7.7 7.7.7.8 7.7.7.9 7.7.8.1 7.7.8.2 7.7.8.3 7.7.8.4 7.7.8.5 7.7.8.67.7.8.7 7.7.8.8 7.7.8.9 7.7.9.1 7.7.9.2 7.7.9.3 7.7.9.4 7.7.9.5 7.7.9.67.7.9.7 7.7.9.8 7.7.9.9 7.8.1.1 7.8.1.2 7.8.1.3 7.8.1.4 7.8.1.5 7.8.1.67.8.1.7 7.8.1.8 7.8.1.9 7.8.2.1 7.8.2.2 7.8.2.3 7.8.2.4 7.8.2.5 7.8.2.67.8.2.7 7.8.2.8 7.8.2.9 7.8.3.1 7.8.3.2 7.8.3.3 7.8.3.4 7.8.3.5 7.8.3.67.8.3.7 7.8.3.8 7.8.3.9 7.8.4.1 7.8.4.2 7.8.4.3 7.8.4.4 7.8.4.5 7.8.4.67.8.4.7 7.8.4.8 7.8.4.9 7.8.5.1 7.8.5.2 7.8.5.3 7.8.5.4 7.8.5.5 7.8.5.67.8.5.7 7.8.5.8 7.8.5.9 7.8.6.1 7.8.6.2 7.8.6.3 7.8.6.4 7.8.6.5 7.8.6.67.8.6.7 7.8.6.8 7.8.6.9 7.8.7.1 7.8.7.2 7.8.7.3 7.8.7.4 7.8.7.5 7.8.7.67.8.7.7 7.8.7.8 7.8.7.9 7.8.8.1 7.8.8.2 7.8.8.3 7.8.8.4 7.8.8.5 7.8.8.67.8.8.7 7.8.8.8 7.8.8.9 7.8.9.1 7.8.9.2 7.8.9.3 7.8.9.4 7.8.9.5 7.8.9.67.8.9.7 7.8.9.8 7.8.9.9 7.9.1.1 7.9.1.2 7.9.1.3 7.9.1.4 7.9.1.5 7.9.1.67.9.1.7 7.9.1.8 7.9.1.9 7.9.2.1 7.9.2.2 7.9.2.3 7.9.2.4 7.9.2.5 7.9.2.67.9.2.7 7.9.2.8 7.9.2.9 7.9.3.1 7.9.3.2 7.9.3.3 7.9.3.4 7.9.3.5 7.9.3.67.9.3.7 7.9.3.8 7.9.3.9 7.9.4.1 7.9.4.2 7.9.4.3 7.9.4.4 7.9.4.5 7.9.4.67.9.4.7 7.9.4.8 7.9.4.9 7.9.5.1 7.9.5.2 7.9.5.3 7.9.5.4 7.9.5.5 7.9.5.67.9.5.7 7.9.5.8 7.9.5.9 7.9.6.1 7.9.6.2 7.9.6.3 7.9.6.4 7.9.6.5 7.9.6.67.9.6.7 7.9.6.8 7.9.6.9 7.9.7.1 7.9.7.2 7.9.7.3 7.9.7.4 7.9.7.5 7.9.7.67.9.7.7 7.9.7.8 7.9.7.9 7.9.8.1 7.9.8.2 7.9.8.3 7.9.8.4 7.9.8.5 7.9.8.67.9.8.7 7.9.8.8 7.9.8.9 7.9.9.1 7.9.9.2 7.9.9.3 7.9.9.4 7.9.9.5 7.9.9.67.9.9.7 7.9.9.8 7.9.9.9 8.1.1.1 8.1.1.2 8.1.1.3 8.1.1.4 8.1.1.5 8.1.1.68.1.1.7 8.1.1.8 8.1.1.9 8.1.2.1 8.1.2.2 8.1.2.3 8.1.2.4 8.1.2.5 8.1.2.68.1.2.7 8.1.2.8 8.1.2.9 8.1.3.1 8.1.3.2 8.1.3.3 8.1.3.4 8.1.3.5 8.1.3.68.1.3.7 8.1.3.8 8.1.3.9 8.1.4.1 8.1.4.2 8.1.4.3 8.1.4.4 8.1.4.5 8.1.4.68.1.4.7 8.1.4.8 8.1.4.9 8.1.5.1 8.1.5.2 8.1.5.3 8.1.5.4 8.1.5.5 8.1.5.68.1.5.7 8.1.5.8 8.1.5.9 8.1.6.1 8.1.6.2 8.1.6.3 8.1.6.4 8.1.6.5 8.1.6.68.1.6.7 8.1.6.8 8.1.6.9 8.1.7.1 8.1.7.2 8.1.7.3 8.1.7.4 8.1.7.5 8.1.7.68.1.7.7 8.1.7.8 8.1.7.9 8.1.8.1 8.1.8.2 8.1.8.3 8.1.8.4 8.1.8.5 8.1.8.68.1.8.7 8.1.8.8 8.1.8.9 8.1.9.1 8.1.9.2 8.1.9.3 8.1.9.4 8.1.9.5 8.1.9.68.1.9.7 8.1.9.8 8.1.9.9 8.2.1.1 8.2.1.2 8.2.1.3 8.2.1.4 8.2.1.5 8.2.1.68.2.1.7 8.2.1.8 8.2.1.9 8.2.2.1 8.2.2.2 8.2.2.3 8.2.2.4 8.2.2.5 8.2.2.68.2.2.7 8.2.2.8 8.2.2.9 8.2.3.1 8.2.3.2 8.2.3.3 8.2.3.4 8.2.3.5 8.2.3.68.2.3.7 8.2.3.8 8.2.3.9 8.2.4.1 8.2.4.2 8.2.4.3 8.2.4.4 8.2.4.5 8.2.4.68.2.4.7 8.2.4.8 8.2.4.9 8.2.5.1 8.2.5.2 8.2.5.3 8.2.5.4 8.2.5.5 8.2.5.68.2.5.7 8.2.5.8 8.2.5.9 8.2.6.1 8.2.6.2 8.2.6.3 8.2.6.4 8.2.6.5 8.2.6.68.2.6.7 8.2.6.8 8.2.6.9 8.2.7.1 8.2.7.2 8.2.7.3 8.2.7.4 8.2.7.5 8.2.7.68.2.7.7 8.2.7.8 8.2.7.9 8.2.8.1 8.2.8.2 8.2.8.3 8.2.8.4 8.2.8.5 8.2.8.68.2.8.7 8.2.8.8 8.2.8.9 8.2.9.1 8.2.9.2 8.2.9.3 8.2.9.4 8.2.9.5 8.2.9.68.2.9.7 8.2.9.8 8.2.9.9 8.3.1.1 8.3.1.2 8.3.1.3 8.3.1.4 8.3.1.5 8.3.1.68.3.1.7 8.3.1.8 8.3.1.9 8.3.2.1 8.3.2.2 8.3.2.3 8.3.2.4 8.3.2.5 8.3.2.68.3.2.7 8.3.2.8 8.3.2.9 8.3.3.1 8.3.3.2 8.3.3.3 8.3.3.4 8.3.3.5 8.3.3.68.3.3.7 8.3.3.8 8.3.3.9 8.3.4.1 8.3.4.2 8.3.4.3 8.3.4.4 8.3.4.5 8.3.4.68.3.4.7 8.3.4.8 8.3.4.9 8.3.5.1 8.3.5.2 8.3.5.3 8.3.5.4 8.3.5.5 8.3.5.68.3.5.7 8.3.5.8 8.3.5.9 8.3.6.1 8.3.6.2 8.3.6.3 8.3.6.4 8.3.6.5 8.3.6.68.3.6.7 8.3.6.8 8.3.6.9 8.3.7.1 8.3.7.2 8.3.7.3 8.3.7.4 8.3.7.5 8.3.7.68.3.7.7 8.3.7.8 8.3.7.9 8.3.8.1 8.3.8.2 8.3.8.3 8.3.8.4 8.3.8.5 8.3.8.68.3.8.7 8.3.8.8 8.3.8.9 8.3.9.1 8.3.9.2 8.3.9.3 8.3.9.4 8.3.9.5 8.3.9.68.3.9.7 8.3.9.8 8.3.9.9 8.4.1.1 8.4.1.2 8.4.1.3 8.4.1.4 8.4.1.5 8.4.1.68.4.1.7 8.4.1.8 8.4.1.9 8.4.2.1 8.4.2.2 8.4.2.3 8.4.2.4 8.4.2.5 8.4.2.68.4.2.7 8.4.2.8 8.4.2.9 8.4.3.1 8.4.3.2 8.4.3.3 8.4.3.4 8.4.3.5 8.4.3.68.4.3.7 8.4.3.8 8.4.3.9 8.4.4.1 8.4.4.2 8.4.4.3 8.4.4.4 8.4.4.5 8.4.4.68.4.4.7 8.4.4.8 8.4.4.9 8.4.5.1 8.4.5.2 8.4.5.3 8.4.5.4 8.4.5.5 8.4.5.68.4.5.7 8.4.5.8 8.4.5.9 8.4.6.1 8.4.6.2 8.4.6.3 8.4.6.4 8.4.6.5 8.4.6.68.4.6.7 8.4.6.8 8.4.6.9 8.4.7.1 8.4.7.2 8.4.7.3 8.4.7.4 8.4.7.5 8.4.7.68.4.7.7 8.4.7.8 8.4.7.9 8.4.8.1 8.4.8.2 8.4.8.3 8.4.8.4 8.4.8.5 8.4.8.68.4.8.7 8.4.8.8 8.4.8.9 8.4.9.1 8.4.9.2 8.4.9.3 8.4.9.4 8.4.9.5 8.4.9.68.4.9.7 8.4.9.8 8.4.9.9 8.5.1.1 8.5.1.2 8.5.1.3 8.5.1.4 8.5.1.5 8.5.1.68.5.1.7 8.5.1.8 8.5.1.9 8.5.2.1 8.5.2.2 8.5.2.3 8.5.2.4 8.5.2.5 8.5.2.68.5.2.7 8.5.2.8 8.5.2.9 8.5.3.1 8.5.3.2 8.5.3.3 8.5.3.4 8.5.3.5 8.5.3.68.5.3.7 8.5.3.8 8.5.3.9 8.5.4.1 8.5.4.2 8.5.4.3 8.5.4.4 8.5.4.5 8.5.4.68.5.4.7 8.5.4.8 8.5.4.9 8.5.5.1 8.5.5.2 8.5.5.3 8.5.5.4 8.5.5.5 8.5.5.68.5.5.7 8.5.5.8 8.5.5.9 8.5.6.1 8.5.6.2 8.5.6.3 8.5.6.4 8.5.6.5 8.5.6.68.5.6.7 8.5.6.8 8.5.6.9 8.5.7.1 8.5.7.2 8.5.7.3 8.5.7.4 8.5.7.5 8.5.7.68.5.7.7 8.5.7.8 8.5.7.9 8.5.8.1 8.5.8.2 8.5.8.3 8.5.8.4 8.5.8.5 8.5.8.68.5.8.7 8.5.8.8 8.5.8.9 8.5.9.1 8.5.9.2 8.5.9.3 8.5.9.4 8.5.9.5 8.5.9.68.5.9.7 8.5.9.8 8.5.9.9 8.6.1.1 8.6.1.2 8.6.1.3 8.6.1.4 8.6.1.5 8.6.1.68.6.1.7 8.6.1.8 8.6.1.9 8.6.2.1 8.6.2.2 8.6.2.3 8.6.2.4 8.6.2.5 8.6.2.68.6.2.7 8.6.2.8 8.6.2.9 8.6.3.1 8.6.3.2 8.6.3.3 8.6.3.4 8.6.3.5 8.6.3.68.6.3.7 8.6.3.8 8.6.3.9 8.6.4.1 8.6.4.2 8.6.4.3 8.6.4.4 8.6.4.5 8.6.4.68.6.4.7 8.6.4.8 8.6.4.9 8.6.5.1 8.6.5.2 8.6.5.3 8.6.5.4 8.6.5.5 8.6.5.68.6.5.7 8.6.5.8 8.6.5.9 8.6.6.1 8.6.6.2 8.6.6.3 8.6.6.4 8.6.6.5 8.6.6.68.6.6.7 8.6.6.8 8.6.6.9 8.6.7.1 8.6.7.2 8.6.7.3 8.6.7.4 8.6.7.5 8.6.7.68.6.7.7 8.6.7.8 8.6.7.9 8.6.8.1 8.6.8.2 8.6.8.3 8.6.8.4 8.6.8.5 8.6.8.68.6.8.7 8.6.8.8 8.6.8.9 8.6.9.1 8.6.9.2 8.6.9.3 8.6.9.4 8.6.9.5 8.6.9.68.6.9.7 8.6.9.8 8.6.9.9 8.7.1.1 8.7.1.2 8.7.1.3 8.7.1.4 8.7.1.5 8.7.1.68.7.1.7 8.7.1.8 8.7.1.9 8.7.2.1 8.7.2.2 8.7.2.3 8.7.2.4 8.7.2.5 8.7.2.68.7.2.7 8.7.2.8 8.7.2.9 8.7.3.1 8.7.3.2 8.7.3.3 8.7.3.4 8.7.3.5 8.7.3.68.7.3.7 8.7.3.8 8.7.3.9 8.7.4.1 8.7.4.2 8.7.4.3 8.7.4.4 8.7.4.5 8.7.4.68.7.4.7 8.7.4.8 8.7.4.9 8.7.5.1 8.7.5.2 8.7.5.3 8.7.5.4 8.7.5.5 8.7.5.68.7.5.7 8.7.5.8 8.7.5.9 8.7.6.1 8.7.6.2 8.7.6.3 8.7.6.4 8.7.6.5 8.7.6.68.7.6.7 8.7.6.8 8.7.6.9 8.7.7.1 8.7.7.2 8.7.7.3 8.7.7.4 8.7.7.5 8.7.7.68.7.7.7 8.7.7.8 8.7.7.9 8.7.8.1 8.7.8.2 8.7.8.3 8.7.8.4 8.7.8.5 8.7.8.68.7.8.7 8.7.8.8 8.7.8.9 8.7.9.1 8.7.9.2 8.7.9.3 8.7.9.4 8.7.9.5 8.7.9.68.7.9.7 8.7.9.8 8.7.9.9 8.8.1.1 8.8.1.2 8.8.1.3 8.8.1.4 8.8.1.5 8.8.1.68.8.1.7 8.8.1.8 8.8.1.9 8.8.2.1 8.8.2.2 8.8.2.3 8.8.2.4 8.8.2.5 8.8.2.68.8.2.7 8.8.2.8 8.8.2.9 8.8.3.1 8.8.3.2 8.8.3.3 8.8.3.4 8.8.3.5 8.8.3.68.8.3.7 8.8.3.8 8.8.3.9 8.8.4.1 8.8.4.2 8.8.4.3 8.8.4.4 8.8.4.5 8.8.4.68.8.4.7 8.8.4.8 8.8.4.9 8.8.5.1 8.8.5.2 8.8.5.3 8.8.5.4 8.8.5.5 8.8.5.68.8.5.7 8.8.5.8 8.8.5.9 8.8.6.1 8.8.6.2 8.8.6.3 8.8.6.4 8.8.6.5 8.8.6.68.8.6.7 8.8.6.8 8.8.6.9 8.8.7.1 8.8.7.2 8.8.7.3 8.8.7.4 8.8.7.5 8.8.7.68.8.7.7 8.8.7.8 8.8.7.9 8.8.8.1 8.8.8.2 8.8.8.3 8.8.8.4 8.8.8.5 8.8.8.68.8.8.7 8.8.8.8 8.8.8.9 8.8.9.1 8.8.9.2 8.8.9.3 8.8.9.4 8.8.9.5 8.8.9.68.8.9.7 8.8.9.8 8.8.9.9 8.9.1.1 8.9.1.2 8.9.1.3 8.9.1.4 8.9.1.5 8.9.1.68.9.1.7 8.9.1.8 8.9.1.9 8.9.2.1 8.9.2.2 8.9.2.3 8.9.2.4 8.9.2.5 8.9.2.68.9.2.7 8.9.2.8 8.9.2.9 8.9.3.1 8.9.3.2 8.9.3.3 8.9.3.4 8.9.3.5 8.9.3.68.9.3.7 8.9.3.8 8.9.3.9 8.9.4.1 8.9.4.2 8.9.4.3 8.9.4.4 8.9.4.5 8.9.4.68.9.4.7 8.9.4.8 8.9.4.9 8.9.5.1 8.9.5.2 8.9.5.3 8.9.5.4 8.9.5.5 8.9.5.68.9.5.7 8.9.5.8 8.9.5.9 8.9.6.1 8.9.6.2 8.9.6.3 8.9.6.4 8.9.6.5 8.9.6.68.9.6.7 8.9.6.8 8.9.6.9 8.9.7.1 8.9.7.2 8.9.7.3 8.9.7.4 8.9.7.5 8.9.7.68.9.7.7 8.9.7.8 8.9.7.9 8.9.8.1 8.9.8.2 8.9.8.3 8.9.8.4 8.9.8.5 8.9.8.68.9.8.7 8.9.8.8 8.9.8.9 8.9.9.1 8.9.9.2 8.9.9.3 8.9.9.4 8.9.9.5 8.9.9.68.9.9.7 8.9.9.8 8.9.9.9 9.1.1.1 9.1.1.2 9.1.1.3 9.1.1.4 9.1.1.5 9.1.1.69.1.1.7 9.1.1.8 9.1.1.9 9.1.2.1 9.1.2.2 9.1.2.3 9.1.2.4 9.1.2.5 9.1.2.69.1.2.7 9.1.2.8 9.1.2.9 9.1.3.1 9.1.3.2 9.1.3.3 9.1.3.4 9.1.3.5 9.1.3.69.1.3.7 9.1.3.8 9.1.3.9 9.1.4.1 9.1.4.2 9.1.4.3 9.1.4.4 9.1.4.5 9.1.4.69.1.4.7 9.1.4.8 9.1.4.9 9.1.5.1 9.1.5.2 9.1.5.3 9.1.5.4 9.1.5.5 9.1.5.69.1.5.7 9.1.5.8 9.1.5.9 9.1.6.1 9.1.6.2 9.1.6.3 9.1.6.4 9.1.6.5 9.1.6.69.1.6.7 9.1.6.8 9.1.6.9 9.1.7.1 9.1.7.2 9.1.7.3 9.1.7.4 9.1.7.5 9.1.7.69.1.7.7 9.1.7.8 9.1.7.9 9.1.8.1 9.1.8.2 9.1.8.3 9.1.8.4 9.1.8.5 9.1.8.69.1.8.7 9.1.8.8 9.1.8.9 9.1.9.1 9.1.9.2 9.1.9.3 9.1.9.4 9.1.9.5 9.1.9.69.1.9.7 9.1.9.8 9.1.9.9 9.2.1.1 9.2.1.2 9.2.1.3 9.2.1.4 9.2.1.5 9.2.1.69.2.1.7 9.2.1.8 9.2.1.9 9.2.2.1 9.2.2.2 9.2.2.3 9.2.2.4 9.2.2.5 9.2.2.69.2.2.7 9.2.2.8 9.2.2.9 9.2.3.1 9.2.3.2 9.2.3.3 9.2.3.4 9.2.3.5 9.2.3.69.2.3.7 9.2.3.8 9.2.3.9 9.2.4.1 9.2.4.2 9.2.4.3 9.2.4.4 9.2.4.5 9.2.4.69.2.4.7 9.2.4.8 9.2.4.9 9.2.5.1 9.2.5.2 9.2.5.3 9.2.5.4 9.2.5.5 9.2.5.69.2.5.7 9.2.5.8 9.2.5.9 9.2.6.1 9.2.6.2 9.2.6.3 9.2.6.4 9.2.6.5 9.2.6.69.2.6.7 9.2.6.8 9.2.6.9 9.2.7.1 9.2.7.2 9.2.7.3 9.2.7.4 9.2.7.5 9.2.7.69.2.7.7 9.2.7.8 9.2.7.9 9.2.8.1 9.2.8.2 9.2.8.3 9.2.8.4 9.2.8.5 9.2.8.69.2.8.7 9.2.8.8 9.2.8.9 9.2.9.1 9.2.9.2 9.2.9.3 9.2.9.4 9.2.9.5 9.2.9.69.2.9.7 9.2.9.8 9.2.9.9 9.3.1.1 9.3.1.2 9.3.1.3 9.3.1.4 9.3.1.5 9.3.1.69.3.1.7 9.3.1.8 9.3.1.9 9.3.2.1 9.3.2.2 9.3.2.3 9.3.2.4 9.3.2.5 9.3.2.69.3.2.7 9.3.2.8 9.3.2.9 9.3.3.1 9.3.3.2 9.3.3.3 9.3.3.4 9.3.3.5 9.3.3.69.3.3.7 9.3.3.8 9.3.3.9 9.3.4.1 9.3.4.2 9.3.4.3 9.3.4.4 9.3.4.5 9.3.4.69.3.4.7 9.3.4.8 9.3.4.9 9.3.5.1 9.3.5.2 9.3.5.3 9.3.5.4 9.3.5.5 9.3.5.69.3.5.7 9.3.5.8 9.3.5.9 9.3.6.1 9.3.6.2 9.3.6.3 9.3.6.4 9.3.6.5 9.3.6.69.3.6.7 9.3.6.8 9.3.6.9 9.3.7.1 9.3.7.2 9.3.7.3 9.3.7.4 9.3.7.5 9.3.7.69.3.7.7 9.3.7.8 9.3.7.9 9.3.8.1 9.3.8.2 9.3.8.3 9.3.8.4 9.3.8.5 9.3.8.69.3.8.7 9.3.8.8 9.3.8.9 9.3.9.1 9.3.9.2 9.3.9.3 9.3.9.4 9.3.9.5 9.3.9.69.3.9.7 9.3.9.8 9.3.9.9 9.4.1.1 9.4.1.2 9.4.1.3 9.4.1.4 9.4.1.5 9.4.1.69.4.1.7 9.4.1.8 9.4.1.9 9.4.2.1 9.4.2.2 9.4.2.3 9.4.2.4 9.4.2.5 9.4.2.69.4.2.7 9.4.2.8 9.4.2.9 9.4.3.1 9.4.3.2 9.4.3.3 9.4.3.4 9.4.3.5 9.4.3.69.4.3.7 9.4.3.8 9.4.3.9 9.4.4.1 9.4.4.2 9.4.4.3 9.4.4.4 9.4.4.5 9.4.4.69.4.4.7 9.4.4.8 9.4.4.9 9.4.5.1 9.4.5.2 9.4.5.3 9.4.5.4 9.4.5.5 9.4.5.69.4.5.7 9.4.5.8 9.4.5.9 9.4.6.1 9.4.6.2 9.4.6.3 9.4.6.4 9.4.6.5 9.4.6.69.4.6.7 9.4.6.8 9.4.6.9 9.4.7.1 9.4.7.2 9.4.7.3 9.4.7.4 9.4.7.5 9.4.7.69.4.7.7 9.4.7.8 9.4.7.9 9.4.8.1 9.4.8.2 9.4.8.3 9.4.8.4 9.4.8.5 9.4.8.69.4.8.7 9.4.8.8 9.4.8.9 9.4.9.1 9.4.9.2 9.4.9.3 9.4.9.4 9.4.9.5 9.4.9.69.4.9.7 9.4.9.8 9.4.9.9 9.5.1.1 9.5.1.2 9.5.1.3 9.5.1.4 9.5.1.5 9.5.1.69.5.1.7 9.5.1.8 9.5.1.9 9.5.2.1 9.5.2.2 9.5.2.3 9.5.2.4 9.5.2.5 9.5.2.69.5.2.7 9.5.2.8 9.5.2.9 9.5.3.1 9.5.3.2 9.5.3.3 9.5.3.4 9.5.3.5 9.5.3.69.5.3.7 9.5.3.8 9.5.3.9 9.5.4.1 9.5.4.2 9.5.4.3 9.5.4.4 9.5.4.5 9.5.4.69.5.4.7 9.5.4.8 9.5.4.9 9.5.5.1 9.5.5.2 9.5.5.3 9.5.5.4 9.5.5.5 9.5.5.69.5.5.7 9.5.5.8 9.5.5.9 9.5.6.1 9.5.6.2 9.5.6.3 9.5.6.4 9.5.6.5 9.5.6.69.5.6.7 9.5.6.8 9.5.6.9 9.5.7.1 9.5.7.2 9.5.7.3 9.5.7.4 9.5.7.5 9.5.7.69.5.7.7 9.5.7.8 9.5.7.9 9.5.8.1 9.5.8.2 9.5.8.3 9.5.8.4 9.5.8.5 9.5.8.69.5.8.7 9.5.8.8 9.5.8.9 9.5.9.1 9.5.9.2 9.5.9.3 9.5.9.4 9.5.9.5 9.5.9.69.5.9.7 9.5.9.8 9.5.9.9 9.6.1.1 9.6.1.2 9.6.1.3 9.6.1.4 9.6.1.5 9.6.1.69.6.1.7 9.6.1.8 9.6.1.9 9.6.2.1 9.6.2.2 9.6.2.3 9.6.2.4 9.6.2.5 9.6.2.69.6.2.7 9.6.2.8 9.6.2.9 9.6.3.1 9.6.3.2 9.6.3.3 9.6.3.4 9.6.3.5 9.6.3.69.6.3.7 9.6.3.8 9.6.3.9 9.6.4.1 9.6.4.2 9.6.4.3 9.6.4.4 9.6.4.5 9.6.4.69.6.4.7 9.6.4.8 9.6.4.9 9.6.5.1 9.6.5.2 9.6.5.3 9.6.5.4 9.6.5.5 9.6.5.69.6.5.7 9.6.5.8 9.6.5.9 9.6.6.1 9.6.6.2 9.6.6.3 9.6.6.4 9.6.6.5 9.6.6.69.6.6.7 9.6.6.8 9.6.6.9 9.6.7.1 9.6.7.2 9.6.7.3 9.6.7.4 9.6.7.5 9.6.7.69.6.7.7 9.6.7.8 9.6.7.9 9.6.8.1 9.6.8.2 9.6.8.3 9.6.8.4 9.6.8.5 9.6.8.69.6.8.7 9.6.8.8 9.6.8.9 9.6.9.1 9.6.9.2 9.6.9.3 9.6.9.4 9.6.9.5 9.6.9.69.6.9.7 9.6.9.8 9.6.9.9 9.7.1.1 9.7.1.2 9.7.1.3 9.7.1.4 9.7.1.5 9.7.1.69.7.1.7 9.7.1.8 9.7.1.9 9.7.2.1 9.7.2.2 9.7.2.3 9.7.2.4 9.7.2.5 9.7.2.69.7.2.7 9.7.2.8 9.7.2.9 9.7.3.1 9.7.3.2 9.7.3.3 9.7.3.4 9.7.3.5 9.7.3.69.7.3.7 9.7.3.8 9.7.3.9 9.7.4.1 9.7.4.2 9.7.4.3 9.7.4.4 9.7.4.5 9.7.4.69.7.4.7 9.7.4.8 9.7.4.9 9.7.5.1 9.7.5.2 9.7.5.3 9.7.5.4 9.7.5.5 9.7.5.69.7.5.7 9.7.5.8 9.7.5.9 9.7.6.1 9.7.6.2 9.7.6.3 9.7.6.4 9.7.6.5 9.7.6.69.7.6.7 9.7.6.8 9.7.6.9 9.7.7.1 9.7.7.2 9.7.7.3 9.7.7.4 9.7.7.5 9.7.7.69.7.7.7 9.7.7.8 9.7.7.9 9.7.8.1 9.7.8.2 9.7.8.3 9.7.8.4 9.7.8.5 9.7.8.69.7.8.7 9.7.8.8 9.7.8.9 9.7.9.1 9.7.9.2 9.7.9.3 9.7.9.4 9.7.9.5 9.7.9.69.7.9.7 9.7.9.8 9.7.9.9 9.8.1.1 9.8.1.2 9.8.1.3 9.8.1.4 9.8.1.5 9.8.1.69.8.1.7 9.8.1.8 9.8.1.9 9.8.2.1 9.8.2.2 9.8.2.3 9.8.2.4 9.8.2.5 9.8.2.69.8.2.7 9.8.2.8 9.8.2.9 9.8.3.1 9.8.3.2 9.8.3.3 9.8.3.4 9.8.3.5 9.8.3.69.8.3.7 9.8.3.8 9.8.3.9 9.8.4.1 9.8.4.2 9.8.4.3 9.8.4.4 9.8.4.5 9.8.4.69.8.4.7 9.8.4.8 9.8.4.9 9.8.5.1 9.8.5.2 9.8.5.3 9.8.5.4 9.8.5.5 9.8.5.69.8.5.7 9.8.5.8 9.8.5.9 9.8.6.1 9.8.6.2 9.8.6.3 9.8.6.4 9.8.6.5 9.8.6.69.8.6.7 9.8.6.8 9.8.6.9 9.8.7.1 9.8.7.2 9.8.7.3 9.8.7.4 9.8.7.5 9.8.7.69.8.7.7 9.8.7.8 9.8.7.9 9.8.8.1 9.8.8.2 9.8.8.3 9.8.8.4 9.8.8.5 9.8.8.69.8.8.7 9.8.8.8 9.8.8.9 9.8.9.1 9.8.9.2 9.8.9.3 9.8.9.4 9.8.9.5 9.8.9.69.8.9.7 9.8.9.8 9.8.9.9 9.9.1.1 9.9.1.2 9.9.1.3 9.9.1.4 9.9.1.5 9.9.1.69.9.1.7 9.9.1.8 9.9.1.9 9.9.2.1 9.9.2.2 9.9.2.3 9.9.2.4 9.9.2.5 9.9.2.69.9.2.7 9.9.2.8 9.9.2.9 9.9.3.1 9.9.3.2 9.9.3.3 9.9.3.4 9.9.3.5 9.9.3.69.9.3.7 9.9.3.8 9.9.3.9 9.9.4.1 9.9.4.2 9.9.4.3 9.9.4.4 9.9.4.5 9.9.4.69.9.4.7 9.9.4.8 9.9.4.9 9.9.5.1 9.9.5.2 9.9.5.3 9.9.5.4 9.9.5.5 9.9.5.69.9.5.7 9.9.5.8 9.9.5.9 9.9.6.1 9.9.6.2 9.9.6.3 9.9.6.4 9.9.6.5 9.9.6.69.9.6.7 9.9.6.8 9.9.6.9 9.9.7.1 9.9.7.2 9.9.7.3 9.9.7.4 9.9.7.5 9.9.7.69.9.7.7 9.9.7.8 9.9.7.9 9.9.8.1 9.9.8.2 9.9.8.3 9.9.8.4 9.9.8.5 9.9.8.69.9.8.7 9.9.8.8 9.9.8.9 9.9.9.1 9.9.9.2 9.9.9.3 9.9.9.4 9.9.9.5 9.9.9.69.9.9.7 9.9.9.8 9.9.9.9

Another group of preferred compounds are named in Table 2 and designatedby numbers assigned to the variables of formula I using the followingconvention: M¹.Y/Y′.V/Z/W. The compounds are shown without depiction ofstereochemistry since the compounds are biologically active as thediastereomeric mixture or as a single stereoisomer. M¹ is a variablethat represents compounds of the formula M-H which have a specifichydroxyl group that is phosphorylated with a groupP(O)[Y—CH(V)CH(Z)CH(W)—Y′] to make compounds of formula I or M¹ is avariable that represents phosphonic acids of the formula M-PO₃ ⁼ whichare transformed to compounds of formula I by replacing two oxygens inthe PO₃ ⁼ group with Y—CH(V)CH(Z)CH(W)—Y′.

The structures for variable M¹ are the same as described above.

Variable Y/Y′

1) Y=NH; Y′=oxygen

2) Y=oxygen; Y′=NH

3) Y=NH; Y′=NH

4) Y=N—CH₃; Y′=oxygen

5) Y=oxygen; Y′=NCH3

6) Y=N—CH2CH3; Y′=oxygen

7) Y=N-phenyl; Y′=oxygen

8) Y=Ni-propyl; Y′=oxygen

9) Y=oxygen; Y′=N—CH2CH3

Variable V/Z/W: Group V/Z/W1

1) V=phenyl; Z=methyl; W=hydrogen

2) V=3,5-dichlorophenyl; Z=methyl; W=hydrogen

3) V=4-pyridyl; Z=methyl; W=hydrogen

4) V=phenyl; Z=methoxy; W=hydrogen

5) V=3,5-dichlorophenyl; Z=methoxy; W=hydrogen

6) V=4-pyridyl; Z=methoxy; W=hydrogen

7) V=phenyl; Z=hydrogen; W=phenyl

8) V=3,5-dichlorophenyl; Z=hydrogen; W=3,5-dichlorophenyl

9) V=4-pyridyl; Z=hydrogen; W=4-pyridyl

Variable V/Z/W: Group V/Z/W2

1) V=phenyl; Z=NHAc; W=hydrogen

2) V=3,5-dichlorophenyl; Z=NHAc; W=hydrogen

3) V=4-pyridyl; Z=NHAc; W=hydrogen

4) V=phenyl; Z=hydrogen; W=methyl

5) V=3,5-dichlorophenyl; Z=hydrogen; W=methyl

6) V=4-pyridyl; Z=hydrogen; W=methyl

7) V=phenyl; Z=hydroxy; W=hydrogen

8) V=3,5-dichlorophenyl; Z=hydroxy; W=hydrogen

9) V=4-pyridyl; Z=hydroxy; W=hydrogen

Variable V/Z/W: Group V/Z/W3

1) V=hydrogen; Z=CH2OH; W=hydrogen

2) V=hydrogen; Z=CH2OC(O)CH3; W=hydrogen

3) V=hydrogen; Z=CH2OC(O)OCH3; W=hydrogen

4) V=methyl; Z=CH2OH; W=hydrogen

5) V=methyl; Z=CH2OC(O)CH3; W=hydrogen

6) V=methyl; Z=CH2OC(O)OCH3; W=hydrogen

7) Z=hydrogen; V and W=—CH2-CH(OH)CH2-

8) Z=hydrogen; V and W=—CH2-CH(OAc)CH2-

9) Z=hydrogen; V and W=—CH2-CH(OCO2CH2CH3)CH2-

Preferred compounds are compounds listed in Table 2 using groups M¹1 andV/Z/W1. For example, compound 1.1.3 represents structure 1 of group M¹1,i.e. 3TC; structure 1 of the variable Y/Y′, i.e. Y=NH and Y′=oxygen;structure 3 of group V/Z/W1, i.e. V=4-pyridyl, Z=methyl and W=hydrogen.The compound 1.1.3. therefore is 3TC with theP(O)(N(H)—CH(4-pyridyl)CH(CH3)CH2O) attached to the primary hydroxyl.

Preferred compounds are also compounds listed in Table 2 using groupsM¹1 and V/Z/W2. Preferred compounds are also compounds listed in Table 2using groups M¹1 and V/Z/W3. Preferred compounds are also compoundslisted in Table 2 using groups M¹2 and V/Z/W1. Preferred compounds arealso compounds listed in Table 2 using groups M¹2 and V/Z/W 2. Preferredcompounds are also compounds listed in Table 2 using groups M¹2 andV/Z/W 3. Preferred compounds are also compounds listed in Table 2 usinggroups M¹3 and V/Z/W1. Preferred compounds are also compounds listed inTable 2 using groups M¹3 and V/Z/W 2. Preferred compounds are alsocompounds listed in Table 2 using groups M¹3 and V/Z/W 3.

TABLE 2 1.1.1 1.1.2 1.1.3 1.1.4 1.1.5 1.1.6 1.1.7 1.1.8 1.1.9 1.2.11.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.2.7 1.2.8 1.2.9 1.3.1 1.3.2 1.3.3 1.3.41.3.5 1.3.6 1.3.7 1.3.8 1.3.9 1.4.1 1.4.2 1.4.3 1.4.4 1.4.5 1.4.6 1.4.71.4.8 1.4.9 1.5.1 1.5.2 1.5.3 1.5.4 1.5.5 1.5.6 1.5.7 1.5.8 1.5.9 1.6.11.6.2 1.6.3 1.6.4 1.6.5 1.6.6 1.6.7 1.6.8 1.6.9 1.7.1 1.7.2 1.7.3 1.7.41.7.5 1.7.6 1.7.7 1.7.8 1.7.9 1.8.1 1.8.2 1.8.3 1.8.4 1.8.5 1.8.6 1.8.71.8.8 1.8.9 1.9.1 1.9.2 1.9.3 1.9.4 1.9.5 1.9.6 1.9.7 1.9.8 1.9.9 2.1.12.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.1.8 2.1.9 2.2.1 2.2.2 2.2.3 2.2.42.2.5 2.2.6 2.2.7 2.2.8 2.2.9 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 2.3.72.3.8 2.3.9 2.4.1 2.4.2 2.4.3 2.4.4 2.4.5 2.4.6 2.4.7 2.4.8 2.4.9 2.5.12.5.2 2.5.3 2.5.4 2.5.5 2.5.6 2.5.7 2.5.8 2.5.9 2.6.1 2.6.2 2.6.3 2.6.42.6.5 2.6.6 2.6.7 2.6.8 2.6.9 2.7.1 2.7.2 2.7.3 2.7.4 2.7.5 2.7.6 2.7.72.7.8 2.7.9 2.8.1 2.8.2 2.8.3 2.8.4 2.8.5 2.8.6 2.8.7 2.8.8 2.8.9 2.9.12.9.2 2.9.3 2.9.4 2.9.5 2.9.6 2.9.7 2.9.8 2.9.9 3.1.1 3.1.2 3.1.3 3.1.43.1.5 3.1.6 3.1.7 3.1.8 3.1.9 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.73.2.8 3.2.9 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7 3.3.8 3.3.9 3.4.13.4.2 3.4.3 3.4.4 3.4.5 3.4.6 3.4.7 3.4.8 3.4.9 3.5.1 3.5.2 3.5.3 3.5.43.5.5 3.5.6 3.5.7 3.5.8 3.5.9 3.6.1 3.6.2 3.6.3 3.6.4 3.6.5 3.6.6 3.6.73.6.8 3.6.9 3.7.1 3.7.2 3.7.3 3.7.4 3.7.5 3.7.6 3.7.7 3.7.8 3.7.9 3.8.13.8.2 3.8.3 3.8.4 3.8.5 3.8.6 3.8.7 3.8.8 3.8.9 3.9.1 3.9.2 3.9.3 3.9.43.9.5 3.9.6 3.9.7 3.9.8 3.9.9 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.1.6 4.1.74.1.8 4.1.9 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8 4.2.9 4.3.14.3.2 4.3.3 4.3.4 4.3.5 4.3.6 4.3.7 4.3.8 4.3.9 4.4.1 4.4.2 4.4.3 4.4.44.4.5 4.4.6 4.4.7 4.4.8 4.4.9 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.5.6 4.5.74.5.8 4.5.9 4.6.1 4.6.2 4.6.3 4.6.4 4.6.5 4.6.6 4.6.7 4.6.8 4.6.9 4.7.14.7.2 4.7.3 4.7.4 4.7.5 4.7.6 4.7.7 4.7.8 4.7.9 4.8.1 4.8.2 4.8.3 4.8.44.8.5 4.8.6 4.8.7 4.8.8 4.8.9 4.9.1 4.9.2 4.9.3 4.9.4 4.9.5 4.9.6 4.9.74.9.8 4.9.9 5.1.1 5.1.2 5.1.3 5.1.4 5.1.5 5.1.6 5.1.7 5.1.8 5.1.9 5.2.15.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.2.7 5.2.8 5.2.9 5.3.1 5.3.2 5.3.3 5.3.45.3.5 5.3.6 5.3.7 5.3.8 5.3.9 5.4.1 5.4.2 5.4.3 5.4.4 5.4.5 5.4.6 5.4.75.4.8 5.4.9 5.5.1 5.5.2 5.5.3 5.5.4 5.5.5 5.5.6 5.5.7 5.5.8 5.5.9 5.6.15.6.2 5.6.3 5.6.4 5.6.5 5.6.6 5.6.7 5.6.8 5.6.9 5.7.1 5.7.2 5.7.3 5.7.45.7.5 5.7.6 5.7.7 5.7.8 5.7.9 5.8.1 5.8.2 5.8.3 5.8.4 5.8.5 5.8.6 5.8.75.8.8 5.8.9 5.9.1 5.9.2 5.9.3 5.9.4 5.9.5 5.9.6 5.9.7 5.9.8 5.9.9 6.1.16.1.2 6.1.3 6.1.4 6.1.5 6.1.6 6.1.7 6.1.8 6.1.9 6.2.1 6.2.2 6.2.3 6.2.46.2.5 6.2.6 6.2.7 6.2.8 6.2.9 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 6.3.6 6.3.76.3.8 6.3.9 6.4.1 6.4.2 6.4.3 6.4.4 6.4.5 6.4.6 6.4.7 6.4.8 6.4.9 6.5.16.5.2 6.5.3 6.5.4 6.5.5 6.5.6 6.5.7 6.5.8 6.5.9 6.6.1 6.6.2 6.6.3 6.6.46.6.5 6.6.6 6.6.7 6.6.8 6.6.9 6.7.1 6.7.2 6.7.3 6.7.4 6.7.5 6.7.6 6.7.76.7.8 6.7.9 6.8.1 6.8.2 6.8.3 6.8.4 6.8.5 6.8.6 6.8.7 6.8.8 6.8.9 6.9.16.9.2 6.9.3 6.9.4 6.9.5 6.9.6 6.9.7 6.9.8 6.9.9 7.1.1 7.1.2 7.1.3 7.1.47.1.5 7.1.6 7.1.7 7.1.8 7.1.9 7.2.1 7.2.2 7.2.3 7.2.4 7.2.5 7.2.6 7.2.77.2.8 7.2.9 7.3.1 7.3.2 7.3.3 7.3.4 7.3.5 7.3.6 7.3.7 7.3.8 7.3.9 7.4.17.4.2 7.4.3 7.4.4 7.4.5 7.4.6 7.4.7 7.4.8 7.4.9 7.5.1 7.5.2 7.5.3 7.5.47.5.5 7.5.6 7.5.7 7.5.8 7.5.9 7.6.1 7.6.2 7.6.3 7.6.4 7.6.5 7.6.6 7.6.77.6.8 7.6.9 7.7.1 7.7.2 7.7.3 7.7.4 7.7.5 7.7.6 7.7.7 7.7.8 7.7.9 7.8.17.8.2 7.8.3 7.8.4 7.8.5 7.8.6 7.8.7 7.8.8 7.8.9 7.9.1 7.9.2 7.9.3 7.9.47.9.5 7.9.6 7.9.7 7.9.8 7.9.9 8.1.1 8.1.2 8.1.3 8.1.4 8.1.5 8.1.6 8.1.78.1.8 8.1.9 8.2.1 8.2.2 8.2.3 8.2.4 8.2.5 8.2.6 8.2.7 8.2.8 8.2.9 8.3.18.3.2 8.3.3 8.3.4 8.3.5 8.3.6 8.3.7 8.3.8 8.3.9 8.4.1 8.4.2 8.4.3 8.4.48.4.5 8.4.6 8.4.7 8.4.8 8.4.9 8.5.1 8.5.2 8.5.3 8.5.4 8.5.5 8.5.6 8.5.78.5.8 8.5.9 8.6.1 8.6.2 8.6.3 8.6.4 8.6.5 8.6.6 8.6.7 8.6.8 8.6.9 8.7.18.7.2 8.7.3 8.7.4 8.7.5 8.7.6 8.7.7 8.7.8 8.7.9 8.8.1 8.8.2 8.8.3 8.8.48.8.5 8.8.6 8.8.7 8.8.8 8.8.9 8.9.1 8.9.2 8.9.3 8.9.4 8.9.5 8.9.6 8.9.78.9.8 8.9.9 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 9.1.6 9.1.7 9.1.8 9.1.9 9.2.19.2.2 9.2.3 9.2.4 9.2.5 9.2.6 9.2.7 9.2.8 9.2.9 9.3.1 9.3.2 9.3.3 9.3.49.3.5 9.3.6 9.3.7 9.3.8 9.3.9 9.4.1 9.4.2 9.4.3 9.4.4 9.4.5 9.4.6 9.4.79.4.8 9.4.9 9.5.1 9.5.2 9.5.3 9.5.4 9.5.5 9.5.6 9.5.7 9.5.8 9.5.9 9.6.19.6.2 9.6.3 9.6.4 9.6.5 9.6.6 9.6.7 9.6.8 9.6.9 9.7.1 9.7.2 9.7.3 9.7.49.7.5 9.7.6 9.7.7 9.7.8 9.7.9 9.8.1 9.8.2 9.8.3 9.8.4 9.8.5 9.8.6 9.8.79.8.8 9.8.9 9.9.1 9.9.2 9.9.3 9.9.4 9.9.5 9.9.6 9.9.7 9.9.8 9.9.9Syntheses of Compounds of Formula I

Synthesis of the compounds encompassed by the present inventionincludes: I). synthesis of prodrugs; and II). synthesis of substituted1,3-hydroxyamines and 1,3-diamines.

I) Syntheses of Prodrugs:

The following procedures on the preparation of prodrugs illustrate thegeneral procedures used to prepare the prodrugs of the invention whichapply to all phosphate, phosphonate- and phosphoramidate containingdrugs. Prodrugs can be introduced at different stages of synthesis of adrug. Most often they are made at a later stage, because of the generalsensitivity of these groups to various reaction conditions. Opticallypure prodrugs containing single isomer at phosphorus center can be madeeither by separation of the diastereomers by a combination of columnchromatography and/or crystallization, or by enantioselective synthesisof chiral activated phosphoramide intermediates.

The preparation of prodrugs is further organized into 1) synthesis viaactivated P(V) intermediates, 2) synthesis via activated P(III)intermediates, 3) synthesis via phosph(on)ate diacid, and 4)miscellaneous methods. Some of the general synthetic methods describedin here are utilized for phosphate and phosphonate ester synthesis.However, these methods are equally applicable in phosphoramidate casealso when the nitrogen in the prodrug moiety is appropriately protectedand/or substituted.

I.1. Synthesis Via Activated P(V) Intermediate:

I.1.a. Synthesis of Activated P(V) Intermediates:

Most preferred synthesis of phosphoramidate nucleotide prodrugs is thereaction of MH hydroxy group with 4-nitrophenyl phosphoramidate (where Lis nitrophenyl) in presence of a strong base such as sodium hydride orlithium hydride. The activated precursor is prepared by reaction ofcommercially available 4-nitrophenyl phosphorochloridate with asubstituted 1,3-aminoalcohol or 1,3-diamine. In general, synthesis ofphosphoroamidate prodrug is achieved by coupling the alcohol with thecorresponding activated phosphoroamidate precursor for example,Chlorophosphoroamidate (L=chloro) addition on to 5′-hydroxy ofnucleoside is a well known method for preparation of phosphoroamidates(Stepanov et al., Tetrahedron Lett., 1989, 30, 5125). The activatedprecursor can be prepared by several well known methods.Chlorophosphoroamidates useful for synthesis of the prodrugs areprepared from the substituted-1,3-propanediamine or 1,3-aminoalcohol(Kirsten, et al, J. Org. Chem., 1997, 62, 6882). Chlorophosphoroamidatemay also be made by oxidation of the corresponding P(III) intermediates(Ozaki, et al, Bull. Chem. Soc. Jpn., 1989, 62, 3869) which are obtainedby reaction of appropriately protected and/or substituted1,3-aminoalcohol or 1,3-diamine with phosphorus trichloride.Alternatively, the chlorophosphoroamidate agent is made by treatingsubstituted-1,3-aminoalcohol or 1,3-diamine with phosphorusoxychloride(Welch, et al., J. Org. Chem., 1990, 55, 5991). Chlorophosphoroamidatespecies may also be generated in situ from corresponding phosphites(Jung, et al., Nucleosides and Nucleotides, 1994, 13, 1597).Phosphoroflouridate intermediate prepared from phosphoroamidic acid mayalso act as a precursor in preparation of cyclic prodrugs (Watanabe etal., Tetrahedron lett., 1988, 29, 5763).

Phosphoramidates (L=NRR′) can also be used as intermediates in the caseof phosphoramidate prodrugs as in the synthesis of phosphate esterswhere the nitrogen is substituted or in the protected form. Monoalkyl ordialkylphosphoramidate (Watanabe, et al, Chem Pharm Bull., 1990, 38,562), triazolophosphoramidate (Yamakage, et al., Tetrahedron, 1989, 45,5459) and pyrrolidinophosphoramidate (Nakayama, et al, J. Am. Chem.Soc., 1990, 112, 6936) are some of the known intermediates used for thepreparation of phosphate esters. Another effective phosphorylatingprocedure is a metal catalyzed addition of cyclic chlorophosphoroamidateadduct of 2-oxazolone. This type of intermediate attains highselectivity in phosphorylation of primary hydroxy group in presence ofsecondary hydroxyl group (Nagamatsu, et al, Tetrahedron Lett., 1987, 28,2375). These agents are obtained by reaction of a chlorophosphoroamidatewith the amine or alternatively by formation of the correspondingphosphoramidite followed by oxidation.

I.b. Synthesis of Chiral Activated Phosphoramidate:

Phosphorylation of an enantiomerically pure substituted 1,3-aminoalcoholor 1,3-diamine with for example, a commercially availablephosphorodichloridate R—OP(O)Cl₂, where RO is a leaving group,preferably aryl substituted with electron withdrawing groups, such as anitro or a chloro, produces two diastereomeric intermediates that can beseparated by a combination of column chromatography and/orcrystallization. Such a method may also be utilized in preparing chiralchlorophosphoroamidate. Chiral phosphoramidate intermediates can beobtained by utilization of optically pure amine as the chiral auxiliary.This type of intermediate are known to undergo stereospecificsubstitution in the phosphate formation (Nakayama, et al. J. Am. Chem.Soc., 1990, 112, 6936). The relative configuration of the phosphorusatom can be determined by comparison of the ³¹P NMR spectra. Thechemical shift of the equatorial phosphoryloxy moiety (trans-isomer) isalways more upfield than the one of the axial isomer (cis-isomer)(Verkade, et al, J. Org. Chem., 1977, 42, 1549).

I.1.c. Synthesis of Prodrugs Using Activated Phosphoramidates:

Coupling of activated phosphoramidates with alcohols (MH) isaccomplished in the presence of an organic base. For example, Chlorophosphoramidates synthesized as described in the earlier section reactwith an alcohol in the presence of a base such as pyridines orN-methylimidazole. In some cases phosphorylation is enhanced by in situgeneration of iodophosphoramidates from chloro (Stomberg, et al.,Nucleosides & Nucleotides., 1987, 5: 815). Phosphoroflouridateintermediates have also been used in phosphorylation reactions in thepresence of a base such as CsF or n-BuLi to generate cyclic prodrugs(Watanabe et al., Tetrahedron lett., 1988, 29, 5763). Phosphoramidateintermediates are shown to couple by transition metal catalysis(Nagamatsu, et al., Tetrahedron Lett., 1987, 28, 2375).

Reaction of the optically pure diastereomer of phosphoramidateintermediate with the hydroxyl of drug in the presence of an acidproduces the optically pure phosphate prodrug by direct S_(N)2(P)reaction (Nakayama, et al., J. Am. Chem. Soc., 1990, 112, 6936).Alternatively, reaction of the optically pure phosphoramidate precursorwith a fluoride source, preferably cesium fluoride or tetrabutylammoniumfluoride, produces the more reactive phosphorofluoridate which reactswith the hydroxyl of the drug to give the optically pure prodrugconfiguration at the phosphorus atom (Ogilvie, et al., J. Am. Chem.Soc., 1977, 99, 1277).

I.2 Synthesis Via Phosphoramidite Intermediate:

I.2.a. Synthesis of Activated P(III) Intermediates:

Phosphorylation of hydroxy and amino groups is achieved using cyclicphosphorylating agents where the agent is at the P(III) oxidation state.One preferred phosphorylating agent is a chloro phosphoramidite (L′chloro). Cyclic chlorophosphoramidites are prepared under mildconditions by reaction of phosphorus trichloride with substituted1,3-aminoalcohols or 1,3-diamines (Wada, et al, Tetrahedron Lett., 1990,31, 6363). Alternatively phosphoramidites can be used as thephosphorylating agent (Beaucage, et al., Tetrahedron, 1993, 49, 6123).Appropriately substituted phoshoramidites can be prepared by reactingcyclic chlorophosphoramidite with N,N-dialkylamine (Perich, et al.,Aust. J. Chem., 1990, 43, 1623. Perich, et al., Synthesis, 1988, 2, 142)or by reaction of commercially available dialkylaminophosphorochloridatewith substituted propyl-1,3-aminoalcohols or 1,3-diamines.

I.2.b. Synthesis of Chiral Activated P(III) Intermediate:

In the cases where unsymmetrical 1,3-aminoalcohols or 1,3-diamines areused, the cyclic phosphoramidites are expected to form a mixture ofchiral isomers. When an optically active pure amino alcohol or diamineis used a chromatographically separable mixture of two stablediastereomers with the leaving group (NRR′) axial and equatorial on thephosphorous atom is expected (Brown et al., Tet., 1990, 46, 4877). Purediasteromers can usually be obtained by chromatographic separation.These intermediates may also be prepared through chirality induction byan optically active amine.

I.2.c. Synthesis of Prodrugs Using Activated Phosphoramidites:

Appropriately substituted chlorophosphoramidites are used tophosphorylate alcohols on nucleosides in the presence of an organic base(e.g., triethylamine, pyridine). Alternatively, the phosphoramidite canbe obtained by coupling the nucleoside with a phosphoramidate in thepresence of a coupling promoter such as tetrazole or benzimidazoliumtriflate (Hayakawa et al., J. Org. Chem., 1996, 61, 7996). Sincecondensation of alcohols with chloro phosphoramidites orphosphoramidites is an S_(N)2(P) reaction, the product is expected tohave an inverted configuration. This allows for the stereoselectivesynthesis of cyclic phosphoramidites of prodrugs. Isomeric mixtures ofphosphorylation reactions can also be equilibrated (e.g. thermalequilibration) to a more thermodynamically stable isomer.

The resulting phosphoramidites of prodrugs are subsequently oxidized tothe corresponding phosphoramidate prodrugs using an oxidant such asmolecular oxygen or t-butylhydroperoxide (Ozaki et al., Tetrahedron.Lett., 1989, 30, 5899). Oxidation of optically pure P(III) intermediateis expected to stereoselectively provide optically active prodrugs(Mikolajczyk, et al., J. Org. Chem., 1978, 43, 2132. Cullis, P. M. J.Chem. Soc., Chem. Commun., 1984, 1510, Verfurth, et al., Chem. Ber.,1991, 129, 1627).

I.3 Synthesis of Phosphoramidate Prodrugs Via Diacids:

Prodrugs of formula I are synthesized by reaction of the correspondingphosphodichloridate and an 1,3-aminoalcohol or 1,3-diamine (Khamnei, etal., J. Med. Chem., 1996, 39:4109). For example, the reaction of aphosphodichloridate with substituted 1,3-aminoalcohols or diamines inthe presence of base (such as pyridine, triethylamine, etc) yieldscompounds of formula I. These conditions can also be applied in thesynthesis of cyclophosphamide analogs where commercially availablebis(2-chloroethyl)phosphoramidic dichloride is coupled withcorresponding substituted 1,3-amino alcohols or 1,3-diamines.

Such reactive dichloridate intermediates, can be prepared from thecorresponding acids and the chlorinating agents e.g. thionyl chloride(Starrett, et al., J. Med. Chem., 1994, 1857), oxalyl chloride (Stowell,et al., Tetrahedron Lett., 1990, 31: 3261), and phosphorus pentachloride(Quast, et al., Synthesis, 1974, 490). Alternatively, thesedichlorophosphonates can also be generated from disilyl esters (Bhongle,et al., Synth. Commun., 1987, 17: 1071) and dialkyl esters (Still, etal., Tetrahedron Lett., 1983, 24: 4405; Patois, et al., Bull. Soc. Chim.Fr., 1993, 130: 485).

Alternatively, acid coupling reagents including, but not limited to,carbodiimides (Alexander, et al., Collect. Czech. Chem. Commun., 1994,59: 1853; Casara, et al., Bioorg. Med. Chem. Lett., 1992, 2: 145;Ohashi, et al., Tetrahedron Lett., 1988, 29: 1189; Hoffman, M.,Synthesis, 1988, 62), andbenzotriazolyloxytris-(dimethylamino)phosphonium salts (Campagne, etal., Tetrahedron Lett., 1993, 34: 6743) are also used in the synthesisof compounds of formula I starting from phosphate or phosphonatediacids.

Chiral phosphonoamidate prodrugs can be synthesized by either resolution(Pogatnic, et al., Tetrahedron Lett., 1997, 38, 3495) or by chiralityinduction (Taapken, et al., Tetrahedron Lett., 1995, 36, 6659; J. Org.Chem., 1998, 63, 8284).

1.4. Miscellaneous Methods:

Phosphorylation of an alcohol is also achieved under Mitsunobu reactionconditions using the cyclic 1′,3′-propanyl phosphoramidic acid in thepresence of triphenylphosphine and diethylazodicarboxylate (Kimura etal., Bull. Chem. Soc. Jpn., 1979, 52, 1191). Furthermore, phosphateprodrugs can be made by conversion of nucleoside to the dichloridateintermediate with phosphoryl chloride in presence of triethylphosphiteand quenching with substituted-1,3-aminoalcohols (Farquhar et al., J.Org. Chem., 1983, 26, 1153).

Phosphorylation can also be achieved by making the mixed anhydride ofthe cyclic diester of phosphoramidic acid and a sulfonyl chloride,preferably 8-quinolinesulfonyl chloride, and reacting the hydroxyl ofthe drug in the presence of a base, preferably methylimidazole (Takaku,et al., J. Org. Chem., 1982, 47, 4937). In addition, starting from achiral cyclic phosphoramidic acid, obtained by chiral resolution(Wynberg, et al., J. Org. Chem., 1985, 50, 4508) is also a source forchiral phosphoramidate prodrugs.

III. Synthesis of Substituted 1,3-Hydroxyamines and 1,3-Diamines:

A large number of synthetic methods are available for the preparation ofsubstituted 1,3-hydroxyamines and 1,3-diamines due to the ubiquitousnature of these functionalities in naturally occurring compounds.Following are some of these methods organized into:

1. synthesis of substituted 1,3-hydroxy amines; 2. synthesis ofsubstituted 1,3-diamines and 3. synthesis of chiral substituted1,3-hydroxyamines and 1,3-diamines.

III.1. Synthesis of Substituted 1,3-Hydroxy Amines:

A general synthetic procedure for 3-aryl-3-hydroxy-propan-1-amine typeof prodrug moiety involves aldol type condensation of aryl esters withalkyl nitriles followed by reduction of resulting substitutedbenzoylacetonitrile (Shih et al., Heterocycles, 1986, 24, 1599). Theprocedure can also be adapted for formation 2-substituted aminopropanolsby using substituted alkylnitrile. In another approach,3-aryl-3-amino-propan-1-ol type of prodrug groups are synthesized fromaryl aldehydes by condensation of malonic acid in presence of ammoniumacetate followed by reduction of resulting substituted b-amino acids.Both these methods enable to introduce wide variety of substitution ofaryl group (Shih, et al., Heterocycles., 1978, 9, 1277). In an alternateapproach, -substituted organolithium compounds of 1-amino-1-aryl ethyldianion generated from styrene type of compounds undergo addition withcarbonyl compounds to give variety of W, W′ substitution by variation ofthe carbonyl compounds (Barluenga, et al., J. Org. Chem., 1979, 44,4798).

Large number of synthetic methods are known for the preparation ofracemic or chiral 1,3-diols. These methods may be utilized in thesynthesis of corresponding substituted 1,3-aminoalcohols or 1,3 diaminesby converting hydroxy functionality to a leaving group and treating withanhydrous ammonia or required primary or secondary amines (Corey, etal., Tetrahedron Lett., 1989, 30, 5207: Gao, et al., J. Org. Chem.,1988, 53, 4081). A similar transformation may also be achieved directlyfrom alcohols in Mitsunobu type of reaction conditions (Hughes, D. L.,Org. React., 1992, 42).

A variety of synthetic methods are known to prepare the following typesof 1,3-diols: a) 1-substituted; b) 2-substituted; and c) 1,2- or1,3-annulated in their racemic or chiral form. Substitution of V, W, Zgroups of formula I, can be introduced or modified either duringsynthesis of aminoalcohols or after the synthesis of prodrugs.

III.1a) 1-Substituted 1,3-Diols:

1,3-Dihydroxy compounds can be synthesized by several well known methodsin literature. Aryl Grignard additions to 1-hydroxy propan-3-al give1-aryl-substituted propan-1,3-diols. This method will enable conversionof various substituted aryl halides to 1-aryl substituted-1,3-propanediols (Coppi, et al., J. Org. Chem., 1988, 53, 911). Aryl halides canalso be used to synthesize 1-substituted propanediols by Heck couplingof 1,3-diox-4-ene followed by reduction and hydrolysis (Sakamoto, etal., Tetrahedron Lett., 1992, 33, 6845). Substituted 1,3-diols can begenerated enanatioselective reduction of vinyl ketone and hydroborationor by kinetic resolution of allylic alcohol. Variety of aromaticaldehydes can be converted to 1-substituted-1,3-diols by vinyl Grignardaddition followed by hydroboration. Substituted aromatic aldehydes arealso utilized by lithium-t-butylacetate addition followed by esterreduction (Turner., J. Org. Chem., 1990, 55 4744). In another method,commercially available cinnamyl alcohols can be converted to epoxyalcohols under catalytic asymmetric epoxidation conditions. These epoxyalcohols are reduced by Red-Al to result in enantiomerically pure1,3-diols (Gao, et al., J. Org. Chem., 1980, 53, 4081). Alternatively,enantiomerically pure 1,3-diols can be obtained by chiral boranereduction of hydroxyethyl aryl ketone derivatives (Ramachandran, et al.,Tetrahedron Lett., 1997, 38 761). Pyridyl, quinoline, isoquinolinepropan-3-ol derivatives can be oxygenated to 1-substituted-1,3-diol byN-oxide formation followed by rearrangement in acetic anhydrideconditions (Yamamoto, et al., Tetrahedron, 1981, 37, 1871). Aldolcondensation is another well described method for synthesis of the1,3-oxygenated functionality (Mukaiyama, Org. React., 1982, 28, 203).Chral substituted diols can also be made by enantioselective reductionof carbonyl compounds, by chiral aldol condensation or by enzymepromoted kinetic resolution.

III.1b) 2-Substituted 1,3-Diols:

Various 2-substituted-1,3-diols can be made from commercially available2-(hydroxymethyl)-1,3-propane diol. Pentaerythritol can be converted totriol via decarboxylation of diacid followed by reduction (Werle, etal., Liebigs. Ann. Chem., 1986, 944) or diol-monocarboxylic acidderivatives can also be obtained by decarboxylation under knownconditions (Iwata, et al., Tetrahedron lett. 1987, 28, 3131). Nitrotriolis also known to give triol by reductive elimination (Latour, et al.,Synthesis, 1987, 8, 742). The triol can be derivatised by monoacetylation or carbonate formation by treatment with alkanoyl chloride,or alkylchloroformate (Greene and Wuts, Protective groups in organicsynthesis, John Wiley, New York, 1990). Aryl substitution can beaffected by oxidation to aldehyde and aryl Grignard additions. Aldehydescan also be converted to substituted amines by reductive aminationreaction.

III.1c) Cyclic-1,3-diols:

Compounds of formula I where V—Z or V—W are fused by four carbons aremade from Cyclohexane derivatives. Commercially availablecis,cis-1,3,5-cyclohexane triol can be used as is or modified asdescribed in case of 2-substituted propan-1,3-diols to give variousanalogues. These modifications can either be made before or after esterformation. Various 1,3-cyclohexane diols can be made by Diels-Aldermethodology using pyrone as diene (Posner, et al., Tetrahedron Lett.,1991, 32, 5295). Cyclohexyl diol derivatives are also made by nitrileoxide-olefin additions (Curran, et al., J. Am. Chem. Soc., 1985, 107,6023). Alternatively, cyclohexyl precursors are also made fromcommercially available quinic acid (Rao, et al., Tetrahedron Lett.,1991, 32, 547.)

III.2. Synthesis of Substituted 1,3-Diamines:

Substituted 1,3-diamines are synthesized starting from variety ofsubstrates. Arylglutaronitriles can be transformed to 1-substituteddiamines by hydrolysis to amide and Hoffman rearrangement conditions(Bertochio, et al., Bull. Soc. Chim. Fr, 1962, 1809). Whereas,malononitrile substitution will enable variety of Z substitution byelectrophile introduction followed by hydride reduction to correspondingdiamines. In another approach, cinnamaldehydes react with hydrazines orsubstituted hydrazines to give corresponding pyrazolines which uponcatalytic hydrogenation result in substituted 1,3-diamines (Weinhardt,et al., J. Med. Chem., 1985, 28, 694). High trans-diastereoselectivityof 1,3-substitution is also attainable by aryl Grignard addition on topyrazolines followed by reduction (Alexakis, et al., J. Org. Chem.,1992, 576, 4563). 1-Aryl-1,3-diaminopropanes are also prepared bydiborane reduction of 3-amino-3-arylacrylonitriles which in turn aremade from nitrile substituted aromatic compounds (Dornow, et al., Chem.Ber., 1949, 82, 254). Reduction of 1,3-diimines obtained fromcorresponding 1,3-carbonyl compounds are another source of 1,3-diamineprodrug moiety which allows a wide variety of activating groups V and/orZ (Barluenga, et al., J. Org. Chem., 1983, 48, 2255).

III.3. Synthesis of Chiral Substituted 1,3-Hydroxyamines and1,3-Diamines:

Enantiomerically pure 3-aryl-3-hydroxypropan-1-amines are synthesized byCBS enantioselective catalytic reaction of β-chloropropiophenonefollowed by displacement of halo group to make secondary or primaryamines as required (Corey, et al., Tetrahedron Lett., 1989, 30, 5207).Chiral 3-aryl-3-amino propan-1-ol type of prodrug moiety may be obtainedby 1,3-dipolar addition of chirally pure olefin and substituted nitroneof arylaldehyde followed by reduction of resulting isoxazolidine(Koizumi, et al., J. Org. Chem., 1982, 47, 4005). Chiral induction in1,3-polar additions to form substituted isoxazolidines is also attainedby chiral phosphine palladium complexes resulting in enatioselectiveformation of δ-amino alcohol (Hori, et al., J. Org. Chem., 1999, 64,5017). Alternatively, optically pure 1-aryl substituted amino alcoholsare obtained by selective ring opening of corresponding chiral epoxyalcohols with desired amines (Canas et al., Tetrahedron Lett., 1991, 32,6931).

Several methods are known for diastereoselective synthesis of1,3-disubstituted aminoalcohols. For example, treatment of(E)-N-cinnamyltrichloroacetamide with hypochlorus acid results intrans-dihydrooxazine which is readily hydrolysed toerythro-β-chloro-α-hydroxy-δ-phenylpropanamine in highdiastereoselectivity (Commercon et al., Tetrahedron Lett., 1990, 31,3871). Diastereoselective formation of 1,3-aminoalcohols is alsoachieved by reductive amination of optically pure 3-hydroxy ketones(Haddad et al., Tetrahedron Lett., 1997, 38, 5981). In an alternateapproach, 3-aminoketones are transformed to 1,3-disubstitutedaminoalcohols in high stereoselectivity by a selective hydride reduction(Barluenga et al., J. Org. Chem., 1992, 57, 1219).

All the above mentioned methods can also be applied to preparecorresponding V—Z or V—W annulated chiral aminoalcohols. Furthermore,such optically pure amino alcohols are also a source to obtain opticallypure diamines by the procedures described earlier in the section.

Formulations

Compounds of the invention are administered orally in a total daily doseof about 0.1 mg/kg/dose to about 100 mg/kg/dose, preferably from about0.3 mg/kg/dose to about 30 mg/kg/dose. The most preferred dose range isfrom 0.5 to 10 mg/kg (approximately 1 to 20 nmoles/kg/dose). The use oftime-release preparations to control the rate of release of the activeingredient may be preferred. The dose may be administered in as manydivided doses as is convenient. When other methods are used (e.g.intravenous administration), compounds are administered to the affectedtissue at a rate from 0.3 to 300 nmol/kg/min, preferably from 3 to 100nmoles/kg/min. Such rates are easily maintained when these compounds areintravenously administered as discussed below.

For the purposes of this invention, the compounds may be administered bya variety of means including orally, parenterally, by inhalation spray,topically, or rectally in formulations containing pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedhere includes subcutaneous, intravenous, intramuscular, andintraarterial injections with a variety of infusion techniques.Intraarterial and intravenous injection as used herein includesadministration through catheters. Oral administration is generallypreferred.

Pharmaceutical compositions containing the active ingredient may be inany form suitable for the intended method of administration. When usedfor oral use for example, tablets, troches, lozenges, aqueous or oilsuspensions, dispersible powders or granules, emulsions, hard or softcapsules, syrups or elixirs may be prepared. Compositions intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents including sweetening agents, flavoringagents, coloring agents and preserving agents, in order to provide apalatable preparation. Tablets containing the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipient which aresuitable for manufacture of tablets are acceptable. These excipients maybe, for example, inert diluents, such as calcium or sodium carbonate,lactose, calcium or sodium phosphate; granulating and disintegratingagents, such as maize starch, or alginic acid; binding agents, such asstarch, gelatin or acacia; and lubricating agents, such as magnesiumstearate, stearic acid or talc. Tablets may be uncoated or may be coatedby known techniques including microencapsulation to delay disintegrationand adsorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate alone orwith a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain 20 to 2000 μmol (approximately 10 to 1000 mg) of active materialcompounded with an appropriate and convenient amount of carrier materialwhich may vary from about 5 to about 95% of the total compositions. Itis preferred that the pharmaceutical composition be prepared whichprovides easily measurable amounts for administration. For example, anaqueous solution intended for intravenous infusion should contain fromabout 0.05 to about 50 μmol (approximately 0.025 to 25 mg) of the activeingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

As noted above, formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in a freeflowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose) surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropyl methylcellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach. This is particularly advantageous with thecompounds of formula I when such compounds are susceptible to acidhydrolysis.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of a drug.

It will be understood, however, that the specific dose level for anyparticular patient will depend on a variety of factors including theactivity of the specific compound employed; the age, body weight,general health, sex and diet of the individual being treated; the timeand route of administration; the rate of excretion; other drugs whichhave previously been administered; and the severity of the particulardisease undergoing therapy, as is well understood by those skilled inthe art.

EXAMPLES

The prodrug compounds of this invention, their intermediates, and theirpreparation can be understood further by the examples which illustratesome of the processes by which these compounds are prepared. Theseexamples should not however be construed as specifically limiting theinvention and variations of the compounds, now known or later developed,are considered to fall within the scope of the present invention ashereinafter claimed.

Compounds of formula I are prepared using procedures detailed in thefollowing examples.

Example 1 General Procedure for Phosphoramidate Prodrugs of PhosphonicAcid Containing Drugs

A suspension of 5-Isobutyl-2-methyl-4-(2-(5-phosphono)furanyl)thiazole(200 mg, 0.6 mmol) in 2.5 mL of thionyl chloride was heated at refluxtemperature for 4 h. The reaction mixture was cooled and evaporated todryness followed by azeotroping with toluene. To the solution of theresulting residue in 4 ml of methylene chloride was added a solution ofamino alcohol (82 mg, 0.54 mmol) and pyridine (0.5 ml, 6 mmol) in 1 mLof methylene chloride.

After stirring at 25° C. for 4 h the reaction was evaporated andcoevaporated with toluene. The crude product was chromatographed byeluting with 10% methanol-dichloromethane to give 52 mg (20.8%) of aless polar isomer and 48 mg (19.2%) of a more polar isomer.

The following compounds were prepared in this manner:

-   1.1:    5-Isobutyl-2-methyl-4-{2-[5-(1-phenyl-1,3-propyl)phosphorimido]-furanyl}thiazole,    less polar isomer. Rf=0.76 in 10% MeOH/CH2Cl2. Anal. Cald. for    C21H25N2O3PS+0.25H2O+0.1HCl: C, 59.40; H, 6.08; N, 6.60. Found: C,    59.42; H, 5.72; N, 6.44.-   1.2:    5-Isobutyl-2-methyl-4-{2-[5-(1-phenyl-1,3-propyl)phosphorimido]-furanyl}thiazole,    more polar isomer. Rf=0.7 in MeOH/CH2Cl2. Anal. Cald. for    C21H25N2O3PS+0.25H2O: C, 59.91; H, 6.1; N, 6.65. Found: C, 60.17; H,    5.81; N, 6.52.-   1.3:    2-Amino-5-isobutyl-4-{2-[5-(1-phenyl-1,3-propyl)phosphoramido]furanyl}thiazole.    Rf=0.53 (silica, 9:1 CH2Cl2/MeOH). Anal. cald. for C20 H24 N3 O3 P    S+0.2CH2Cl2+0.25H2O: C, 55.27; H, 5.72; N, 9.57. Found: C, 55.03; H,    5.42; N, 9.37.-   1.4:    2-Amino-5-isobutyl-4-{2-[5-(1-phenyl-1,3-propyl)phosphoramido]furanyl}thiazole,    less polar isomer. Rf=0.57 (silica, 9:1 CH2Cl2/MeOH). Anal. cald.    for C20 H24 N3 O3 P S+0.15 CH2Cl2: C, 56.26; H, 5.69; N, 9.77.    Found: C, 56.36; H, 5.46; N, 9.59.-   1.5:    2-Amino-5-methylthio-4-{2-[5-(1-phenyl-1,3-propyl)phosphoramido-]furanyl}thiazole,    less polar isomer. Rf=0.59 (silica, 9:1 CH2Cl2/MeOH). Anal. cald.    for C17 H18 N3 O3 P S2+0.4HCl: C, 48.38; H, 4.39; N, 9.96. Found: C,    48.47; H, 4.21; N, 9.96.-   1.6:    2-Amino-5-methylthio-4-{2-[5-(1-phenyl-1,3-propyl)phosphoramido]furanyl}thiazole,    more polar isomer.

Rf=0.56 (silica, 9:1 CH2Cl2/MeOH). Anal. cald. for C17 H18 N3 O3 P S2:C, 50.11; H, 4.45; N, 10.31. Found: C, 49.84; H, 4.19; N, 10.13.

-   1.7:    2-Amino-5-methylthio-4-{2-[5-(N-methyl-1-phenyl-1,3-propyl)phos-phoramido]furanyl}thiazole.    Rf=0.56 (silica, 9:1 CH2Cl2/MeOH). Anal. cald. for C18 H20 N3 O3 P    S2+0.25HCl C: 50.21; H, 4.74; N, 9.76. Found: C, 50.31; H, 4.46; N,    9.79.-   1.8:    2-Amino-5-isobutyl-4-{2-[5-(1-phenyl-1,3-propyl)-N-acetylphosph-oramido]furanyl}thiazole.    Rf=0.62 (silica, 9:1 CH2Cl2/MeOH). Anal. cald. for C22 H26 N3 O4 P    S+1.25H2O: C, 54.82; H, 5.96; N, 8.72. Found: C, 55.09; H, 5.99; N,    8.39.-   1.9:    2-Amino-5-isobutyl-4-{2-[5-(1-[2,4-dichlorophenyl]-propan-1,3-yl)phosphoramido]furanyl}thiazole.    mp 235-237 C (decomp). Rf=0.35 (silica, 3:2 EtOAc/CH2Cl2). Anal.    cald. for C20 H22 C12 N3 O3 P S: C, 49.39; H, 4.56; N, 8.64. Found:    C, 49.04; H, 4.51; N, 8.37.-   1.10: 188-189    9-{2-[1-phenyl-1-aza-3-oxa-2-phosphorinanyl-2-methyloxy]ethyl}adenine.    mp 188-189 C. Rf=0.25 (silica, 9:1 CHCl2/MeOH). Anal. cald. for C17    H21 N6 O3 P+0.5H2O: C, 51.38; H, 5.58; N, 21.15. Found: C, 51.05; H,    5.28; N, 20.77.-   1.11:    2-Amino-5-isobutyl-4-{2-[5-(1-(3-chlorophenyl)-propan-1,3-yl)phosphoradia-mido]furanyl}thiazole.    Light yellow solid; Rf=0.45 (Silica, 5:95 Methanol/dichloromethane);    ¹H NMR (200 MHz, DMSO-d6) δ 7.35 (m, 4H); 7.1 (m, 1H), 6.6 (m, 1H);    5.55 (m, 1H), 2.8 (d, 2H), 0.9 (d, 6H).

Example 2 Step A

A solution of 3-amino-3-phenyl-1-propanol (1 g, 6.6 mmol) andtriethylamine (3 ml, 21.8 mmol) in tetrahydrofuran (60 ml) was addeddropwise over 20 minutes into a solution of 4-nitrophenylphosphorodichloridate in tetrahydrofuran at 0° C. A white precipitatequickly forms. The yellow heterogeneous mixture was stirred at 0° C. for1 hour then warmed slowly to rt. After stirring at rt for 60 hours, theprecipitate was dissolved with water (40 ml). The clear yellow solutionwas concentrated to ca 40 ml. The resulting mixture was diluted with asaturated aqueous solution of sodium bicarbonate and extracted twicewith ethyl acetate. The combined organic extracts were washed with asaturated aqueous solution of sodium bicarbonate and brine. The aqueouswashes were back extracted and the organic extracts combined. Thecombined organic extracts were dried over sodium sulfate, concentratedand purified by column chromatography (silica gel, hexanes/ethylacetate) to give fast eluting trans isomer (995 mg, 45%) and sloweluting cis isomer (1 g, 45%) and);

Step B

A mixture of 2′,3′-dideoxy-adenosine (50 mg, 0.21 mmol), cesium fluoride(323 mg, 2.1 mmol) and fast eluting(4-Nitrophenoxy)-4-phenyl-1,3,2-oxazaphosphorinan-2-one (142 mg, 0.4mmol) in tent-butanol was heated at 80° C. After 48 hours, more(4-Nitrophenoxy)-4-phenyl-1,3,2-oxazaphosphorinan-2-one (70 mg) wasadded and stirring was continued at 80° C. After 5 days, the cooledyellow heterogeneous mixture was diluted with 50/50methanol/dichloromethane, filtered through a pad of silica and rinsedwith 50/50 methanol/dichloromethane. The combined filtrates wereconcentrated and the residue was purified by column chromatography(silica gel, methanol/dichloromethane) to give the prodrug which was recrystallized from dichloromethane and hexanes: 42 mg (47% yield).

The following compounds are prepared in this manner:

-   2.1    2′,3′-Dideoxy-5′-O-(2-oxo-4-phenyl-1,3,2-oxazaphosphorin-2-yl)-adenosine.    mp 119-122. Rf=0.25 (silica gel, 1/9 methanol/dichloromethane).    Anal. Cald. for C₁₉H₂₃N₆O₄P+0.9 mol dichloromethane: C, 47.16; H,    4.93; N, 16.58. Found: C, 47.03; H, 4.98; N, 16.61.-   2.2    3′-Azido-3′-deoxy-5′-O-(4-phenyl-2-oxo-1,3,2-oxazaphosphorin-2-y-l)-thymidine.    mp 85-105. Rf=0.5 (silica gel, 1/9 methanol/dichloromethane). Anal.    cald. for C₁₉H₂₃N₆O₆P: C, 49.35; H, 5.01; N, 18.17. Found: C, 49.32;    H, 5.17; N, 17.89.

Example 3 Step A

Same as step A of example 2.

Step B

Lithium hydride (15 mg, 1.52 mmol) was added to a solution of9-β-D-arabinofuranosyl-adenine (100 mg, 0.37 mmol) and fast eluting(4-nitrophenoxy)-4-phenyl-1,3,2-oxazaphosphorinan-2-one (250 mg, 0.74mmol) in dimethylformamide at room temperature. After 6 hours the yellowheterogeneous mixture was neutralized with acetic acid. The clear yellowsolution was concentrated and the residue was purified by columnchromatography (silica gel, methanol/dichloromethane). The isolatedproduct was further purified by preparative TLC and recrystallized fromethanol to give the prodrug: 18 mg (11% yield);

The following compound is prepared in this manner:

-   3.1    5′-O-(2-oxo-4-Phenyl-1,3,2-oxazaphosphorin-2-yl)-9-β-D-arabinofuranosyl-adenine.    mp>250. Rf=0.25 (silica gel, 2/8 methanol/dichloromethane). Anal.    cald. for C₁₉H₂₃N₆O₆P: C, 49.35; H, 5.01; N, 18.17. Found: C, 49.22;    H, 4.89; N, 18.03.

The following compounds are made in a similar manner with the exceptionthat the reactions were neutralized with 1N solution of hydrochloricacid during work up.

-   3.2    9-[(2-(4-Phenyl-2-oxo-1,3,2-oxazaphosphorinan-2-oxy)-ethoxy)methyl]-guanin:    mp 151-165; Rf=0.2 (silica gel, 2/8 methanol/dichloromethane); Anal.    cald. for C₁₇H₂₁N₆O₅P+2H2O: C, 44.74; H, 5.52; N, 18.41. Found: C,    44.45; H, 5.40; N, 18.14.-   3.3    2′-Deoxy-5-fluoro-5′-O-(4-phenyl-2-oxo-1,3,2-oxazaphosphorin-2-yl)-uridine.    mp 188-195 Dec. Rf=0.25 (silica gel, 1/9 methanol/dichloromethane).    MS cald. for C₁₈H₂₁FN₃O₇P+Na⁺: 464. Found: 464; MS cald. for    C₁₈H₂₁FN₃O₇P—H: 440. Found: 440.

Example 4 General Procedure for Formation of Nucleotide Prodrugs byP(III) Intermediates

To a solution of 1(aryl)-1-(1-alkyl or acyl amino) propanol (1 mmol) indichloromethane (10 mL) is added phosphorus trichloride at 0° C. Thereaction is warmed to room temperature and allowed to stir for 3 h.Reaction mixture is concentrated, azeotroped with toluene (2×10 mL) anddried. Crude chlorophosphoramidite is used in the next step withoutfurther purification.

To a solution of nucleoside (1 mmol) in DMF (10 mL) is addeddiisopropylethylamine (2 mmol) at −40° C. To this mixture is added crudecyclic chlorophoramidite (1 mmole) in 2 mL of DMF. The mixture is warmedto room temperature and stirred for 2 h. The reaction is cooled back to−40° C. and 5-6M t-butylhydroperoxide in decane (2 mmol) is added andleft at room temperature. After overnight stirring, the reaction isconcentrated and crude mixture is chromatographed.

Example 5 General Procedure for Preparation of 3-Aryl-3-Aminoprolpanolsfrom Aryl Aldehydes

-   (Shih, et al., Heterocycles, 1978, 9, 1277)    Step A:

A mixture of an appropriate aryl aldehyde (10 mmol), malonic acid (10mmol), and ammonium acetate (10 mmol) in 50 mL of 95% ethanol is heatedin a water bath at 80° C. Carbon dioxide begins to evolve at 55° C. andthe reaction is complete in 5-7 h. The white solid is collected bysuction and recrystallized from aqueous ethanol to obtain the3-aryl-3-aminopropionic acid.

Step B:

To a suspension of lithium aluminum hydride (50 mmol) in anhydroustetrahydrofuran (40 mL) is added a 3-aryl-3-aminopropionic acid (20mmol) with cooling and stirring. The mixture is refluxed for 3 h,allowed to stand overnight, and treated with moist ether and then withwater to decompose the excess of lithium aluminum hydride. The organiclayer is decanted, and the material left in the flask is extracted withhot ethylacetate (3×50 mL). The organic solutions are combined androtary evaporated. The residue is vacuum distilled or recrystallized toobtain 3-aryl-3-aminopropanol.

Example 6 General Procedure for Preparation of3-Aryl-3-Hydroxypropylamines from Epoxy Cinnamyl Alcohols

-   (Gao, et al., J. Org. Chem., 1988, 53, 4084)    Step A:

To a solution of commercial (−)-(2S,3S)-2,3-epoxycinnamyl alcohol (10.0mmol) in dimethoxyethane (50 mL) is added a 3.4 M solution of sodiumbis(2-methoxyethoxy)aluminum hydride (Red-Al) in toluene (10.5 mmol)dropwise under nitrogen at 0° C. After stirring at room temperature for3 h, the solution is diluted with ether and quenched with 5% HClsolution. After further stirring at room temperature for 30 min, thewhite precipitate formed is removed by filtration and boiled with ethylacetate and filtered again. The combined organic extracts are dried withmagnesium sulfate and concentrated to give(R)-3-phenyl-1,3-dihydroxypropane.

Step B:

To a solution of (R)-3-phenyl-1,3-dihydroxypropane (17.8 mmol) andtriethylamine (25.6 mmol) in ether (90 mL) is added dropwise MsCl (18.7mmol) under nitrogen at −10° C. After stirring at −10 to 0° C. for 3 h,the mixture is poured into ice water (30 mL), organic layer was washedwith 20% H₂SO₄, saturated aqueous NaHCO₃, and brine, and dried overmagnesium sulfate. The crude product is purified by chromatography on asilica gel column.

Step C:

A solution of (R)-3-phenyl-3-(hydroxy)-propyl methanesulfonate (3 mmol)and methylamine (10 mL, 40% in water) in THF (10 mL) is heated at 65° C.for 3 h. After cooling, the solution is diluted with ether, washed withsaturated aqueous sodium bicarbonate and brine, and dried with anhydrouspotassium carbonate. Concentration of extract provides(R)-3-phenyl-3-(hydroxy)-propyl amine.

Example 7 General Procedure for Preparation of 3-Aryl-Propylenediaminesfrom Cinnamyl Aldehydes

-   (Weinhardt, et al., J. Med. Chem., 1985, 28, 694)    Step A:

To a solution of cinnamaldehyde (10 mmol) in 100 mL of EtOH is addedsubstituted hydrazine (10 mmol). The reaction is stirred at roomtemperature for 45 min. Solvents are removed on a rotatory evaporatorand saturated NaHCO₃ was added to the residue. The crude product isextracted into ether. The organic layer is washed, dried and evaporated.Crude products from chromatography results in a pure pyrazoline adduct.

Step B:

A solution of 3-phenylpyrazoline (10 mmol) in 50 mL of AcOH and 10 mL of10% HCl is hydrogenated at atmospheric pressure over 500 mg of 5% Pt/C.The reaction is stirred under hydrogen for 6 h. The catalyst is removedby filtration and the filtrate is concentrated. The crude product ispurified by column to give pure 3-Aryl-propylenediamines derivative.

Example 8 General Procedure for 1,3-Amino Alcohols for 2-SubstitutedProdrugs (V, W and W′=H and Z=—CHR²OH)

Step A:

To a solution of commercially available 2-(hydroxymethyl)-1,3-propanediol (1 mmol) in pyridine (5 mL) is added methanesulfonyl chloride (1mmol) and stirred at room temperature until the reaction is complete.The mixture is concentrated, extracted and product is purified by columnchromatography.

Step B:

A solution of 2-(methylsulfonyloxymethylene)-1,3-propane diol (1 mmol)and ammonium hydroxide (10 mL, 30% in water) in THF (10 mL) is heated at100° C. in a sealed reactor. After cooling, the mixture is concentrated,extracted and product is purified by column chromatography to give2-substituted-1,3-amino alcohol. Appropriate amines may be used in stepB to vary —NR⁶ substitution. 2-substituted-1,3-diamines may also besynthesized using Step A and B from 2-(hydroxymethyl)-1,3-propane diolusing appropriate stoichiometry of reagents.

Step C:

Prodrugs of 2-substituted-1,3-amino alcohols or2-substituted-1,3-diamines are synthesized by following couplingprocedures as described in example 1 or example 2 (steps A and B) orexample 3 (step B) or example 4 depending on the parent compound.

Example 9 General Procedure for Cyclic 1,3-Amino Alcohols (Together Vand W are Connected)

Step A:

Commercially available cis,cis-1,3,5-cyclohexane triol is converted to amono mesylate as described in example 8, step A.

Step B:

cis,cis-1-(methylsulfonyloxy)cyclohexane-3,5-diol is transformed tocorresponding amino alcohol as described in example 8, step B.Appropriate amines may be used in step B to vary —NR⁶ substitution.1-Hydroxy-3,5-diamino cyclohexane may also be synthesized using Step Aand B from cis, cis-1,3,5-cyclohexane triol using appropriatestoichiometry of reagents.

Step C:

Prodrugs of cyclohexyl-1,3-amino alcohols or 1,3-diamines aresynthesized by following procedures as described in example 1 or example2 (steps A and B) or example 3 (step B) or example 4 depending on theparent compound.

Example 10 Preparation of 2-substituted4-[2′-(5′-phosphono)furanyl]thiazoles

Step A:

A solution of furan (1.3 mmole) in toluene was treated with 4-methylpentanoic acid (1 mmole), trifluoroacetic anhydride (1.2 mmole) andboron trifluoride etherate (0.1 mmole) at 56° C. for 3.5 h. The cooledreaction mixture was quenched with aqueous sodium bicarbonate (1.9mmole), filtered through a celite pad. Extraction, evaporation anddistillation gave 2-[(4-methyl-1-oxo)pentyl]furan as a brown oil (bp65-77° C., 0.1 mmHg).

Step B:

A solution of 2-[(4-methyl-1-oxo)pentyl]furan (1 mmole) in benzene wastreated with ethylene glycol (2.1 mmole) and p-toluenesulfonic acid(0.05 mmole) at reflux for 60 h while removing water via a Dean-Starktrap. Triethyl orthoformate (0.6 mmole) was added and resulting mixturewas heated at reflux for an additional hour. Extraction and evaporationgave 2-(2-furanyl)-2-[(3-methyl)butyl]-1,3-dioxolane as an orangeliquid.

Step C:

A solution of 2-(2-furanyl)-2-[(3-methyl)butyl]-1,3-dioxolane (1 mmole)in THF was treated with TMEDA (1 mmole) and nBuLi (1.1 mmole) at −45°C., and the resulting reaction mixture was stirred at −5 to 0° C. for 1h. The resulting reaction mixture was cooled to −45° C., and cannulatedinto a solution of diethyl chlorophosphate in THF at −45° C. Thereaction mixture was gradually warmed to ambient temperature over 1.25h. Extraction and evaporation gave2-[2-(5-diethylphosphono)furanyl]-2-[(3-methyl)butyl]-1,3-dioxolane as adark oil.

Step D:

A solution of2-[2-(5-diethylphosphono)furanyl]-2-[(3-methyl)butyl]-1,3-dioxolane (1mmole) in methanol was treated with 1 N hydrochloric acid (0.2 mmole) at60° C. for 18 h. Extraction and distillation gave5-diethylphosphono-2-[(4-methyl-1-oxo)pentyl]furan as a light orange oil(bp 152-156° C., 0.1 mmHg).

Step E:

A solution of compound gave5-diethylphosphono-2-[(4-methyl-1-oxo)pentyl]furan (1 mmole) in ethanolwas treated with copper (II) bromide (2.2 mmole) at reflux for 3 h. Thecooled reaction mixture was filtered and the filtrate was evaporated todryness. The resulting dark oil was purified by chromatography to give5-diethylphosphono-2-[(2-bromo-4-methyl-1-oxo)pentyl]furan as an orangeoil.

Step F:

A solution of 5-diethylphosphono-2-[(2-bromo-4-methyl-1-oxo)pentyl]furan(1 mmole) and thiourea (2 mmole) in ethanol was heated at reflux for 2h. The cooled reaction mixture was evaporated to dryness and theresulting yellow foam was suspended in saturated sodium bicarbonate andwater (pH=8). The resulting yellow solid was collected throughfiltration to give2-amino-5-isobutyl-4-[2-(5-diethylphosphono)furanyl]thiazole.

Step G:

A solution of2-amino-5-isobutyl-4-[2-(5-diethylphosphono)-furanyl]thiazole (1 mmole)in methylene chloride was treated with bromotrimethylsilane (10 mmole)at 25° C. for 8 h. The reaction mixture was evaporated to dryness andthe residue was suspended in water. The resulting solid was collectedthrough filtration to give2-amino-5-isobutyl-4-[2-(5-phosphono)furanyl]thiazole as an off-whitesolid.

-   10.1 2-amino-5-isobutyl-4-[2′-(5′-phosphono)furanyl]thiazole    mp>250° C. Anal. calcd. for C₁₁H₁₅N₂O₄PS+1.25HBr: C, 32.75; H, 4.06;    N, 6.94. Found: C, 32.39; H, 4.33; N, 7.18.

The following compounds were prepared according to similar procedure:

-   10.2 2-Methyl-5-isobutyl-4-[2′-(5′-phosphono)furanyl]thiazole. Anal.    calcd. for C₁₂H₁₆NO₄PS+HBr+0.1CH₂Cl₂: C, 37.20; H, 4.44; N, 3.58.    Found: C, 37.24; H, 4.56; N, 3.30.-   10.3 2-Amino-5-methylthio-4-[2′-(5′-phosphono)furanyl]thiazole. mp    181-184° C. Anal. calcd. for C₈H₉N₂O₄PS2+0.4H₂O: C, 32.08; H, 3.30;    N, 9.35.

Found: C, 32.09; H, 3.31; N, 9.15.

Example 11 General Procedure for the Synthesis of CyclophosphamideAnalogs

-   (Boyd, et al., J. Med. Chem. 1980, 23, 372)

A solution of bis(2-chloroethyl)phosphoramidic dichloride (10 mmol) inethyl acetate (100 mL) is added dropwise to a solution of commercial 3methylamino-1-phenyl-1-propanol (10 mmol) and triethylamine (20 mmol) inethyl acetate (100 mL) at rt. After stirring at rt for 3 days, the saltsare filtered off and the combined filtrates are concentrated undervacuum. The crude product is purified by column chromatography onsilica.

Example 12 Phosphoramide Prodrugs of 1-Methylamino-1-phenyl-3-propanol

Step A:

The reagentN-methyl-2-(4-nitrophenoxy)-2-oxo-6-phenyl-1,3,2-oxazaphosphorinane wasmade using the same procedure as the one described for step A of example2:

Fast eluting isomer: White solid, mp 135° C.; Rf=0.4 (Silica, Ethylacetate/Hexanes 1/1). Slow eluting isomer: White solid, mp 128-129;Rf=0.15 (Silica, Ethyl acetate/Hexanes 1/1).

Step B:

The coupling step with the nucleoside was accomplished in a mannersimilar to the one described in step B of example 3 except that thereaction mixture was stirred at 100.degree. C. for 5 hours.

The following compound was prepared in this manner:

-   12.1:    9-[(2-(N-Methyl-2-oxo-6-phenyl-1,3,2-oxazaphosphorin-2-oxy)-ethoxy)methyl]-guanine:    pale yellow amorphous solid; Rf=0.45 (Silica,    Methanol/dichloromethane 2/8); ¹H NMR (200 MHz, DMSO d₆) δ 7.8 (bs,    1H); 7.3 (bs, 5H), 6.75 (bs, 2H, exchangeable with D₂O); 5.35 (bs,    3H).

The following compound was prepared in this manner:

-   12.2:    5-(N-Methyl-2-oxo-6-phenyl-1,3,2-oxazaphosphorin-2-yl)-9-β-D-arabino-furanosyladenine:    Off white amorphous solid; Rf=0.2 (Silica, Methanol/dichloromethane    1/9); ¹H NMR (200 MHz, DMSO d₆) δ 7.4-7.1 (m, 5H); 5.75 (s, 1H),    5.25 (m, 1H); 4.8 (m, 1H).

Example 13 Phosphoramide Prodrugs of 3-amino-1-hydroxy-1-phenyl propane

Step A:

A mixture of 3-phenyl-3-hydroxypropylmethanesulfonate (1.71 g, Gao, etal., J. Org. Chem., 1988, 53, 4081), tetrahydrofuran (10 mL) and 28%ammonium hydroxide (30 mL) was heated in a bomb at 65° C. for 16 hours.After cooling, the volatiles were eliminated to leave the aqueoussolution which was extracted with ether. The aqueous layer wasconcentrated under reduced pressure, azeotroped twice with anhydrousacetonitrile and dried under vacuum to give 3-amino-1-phenyl-1-propanol(1.04 g) as a white solid: mp 92-94° C., Rf=0.1 (silica, methanol/ethylacetate 1/1).

Step B:

The reagent 2-(4-nitrophenoxy)-2-oxo-6-phenyl-1,3,2-oxazaphosphorinane(13.1) was made using the same procedure as the one described for step Aof example 2: Fast eluting isomer: Rf=0.6 (Silica, Ethyl acetate), ¹HNMR (200 MHz, DMSO d₆) δ 8.22 (m, 2H); 7.48 (m, 2H), 7.35 (m, 5H); 5.52(m, 1H).

Step C:

The coupling step with the nucleoside is accomplished in a mannersimilar to the one described in step B of example 3.

The following compounds are made in this manner:

-   9-[(2-(N-Methyl-2-oxo-6-phenyl-1,3,2-oxazaphosphorin-2-oxy)-ethoxy)methyl]-guanine;-   5-(N-Methyl-2-oxo-6-phenyl-1,3,2-oxazaphosphorin-2-yl)-9-β-D-arabinofuranosyladenine.

Examples of use of the method of the invention includes the following.It will be understood that these examples are exemplary and that themethod of the invention is not limited solely to these examples.

For the purposes of clarity and brevity, chemical compounds are referredto as synthetic example numbers in the biological examples below.

BIOLOGICAL EXAMPLES Example A Chemical Stability of an AntidiabeticPhosphoramidate Prodrug

Methods: Aliquots of a 10 μg/mL solution of Compound 1.3 in potassiumphosphate buffers at pH 3 and 7 (room temperature) were sampled hourlyfor 12 hours. Samples were analyzed by reverse phase HPLC with use of aBeckman Ultrasphere C8 column (4.6×250 mm). The column was equilibratedand eluted with a gradient from 50 mM sodium phosphate pH 5.5 to 80%acetonitrile at a flow rate of 1.0 mL/min. Detection was at 254 nm.Under these conditions, Compound 1.3 was separated from2-Amino-5-isobutyl-4-[2-(5-phosphonomonoamide)furanyl]thiazole, and from2-Amino-5-isobutyl-4-(2-furanyl)thiazole (retention times=17, 15.8, and15.6 min., respectively).

Results: The half-life of Compound 1.3 at pH 3.0 was 3.8 h; prodrugdecomposed to an unidentified metabolite at this pH. Compound 1.3 wasfully stable at pH 7.0; there was no evidence of decompositionthroughout the 12-hour incubation.

Example B Stability of Phosphoramidate Prodrugs to Esterases,Phosphatases, Adenosine Deaminase, and Plasma

Methods: Carboxylesterase (porcine liver) and alkaline phosphatase (calfintestinal mucose) are purchased from Sigma Chemical Co. (St. Louis,Mo.). Carboxylesterase activity is measured in 0.1 M Tris-HCl buffer atpH 8.0. Activity towards p-nitrophenyl acetate, a known substrate andpositive control in the reactions is measured as described for exampleby Matsushima M., et al. [FEBS Lett. (1991)293(1-2): 37-41]. Alkalinephosphatase activity is measured in a 0.1 M diethanolamine buffer, pH9.8, containing 0.5 mM MgCl₂. Activity towards p-nitrophenyl phosphate,a known substrate and positive control in the reactions, is measured asdescribed [e.g. Brenna O., et at (1975) Biochem J. 151(2): 291-6].Adenosine deaminase activity is measured as described by e.g. Gustin NC, et al. [Anal. Biochem. (1976) 71: 527-532]. Adenosine is used as apositive control in these reactions. Plasma is prepared bycentrifugation from fresh, heparinized rat or human blood. Prodrugs areincubated at a concentration of, for example, 25 μM in appropriatereaction mixtures containing carboxylesterase, alkaline phosphatase,adenosine deaminase, or rat or human plasma. In the case of theesterase, phosphatase, and adenosine deaminase assays, parallelreactions are run with known substrates of the enzymes as described.

Aliqouts are removed from the reaction mixture at various time pointsand the reaction stopped by addition of methanol to 60%. Followingcentrifugation and filtration, the methanolic aliquots are analyzed forgeneration of MPO₂—(NHR⁶⁻) or other metabolites by standard reversephase, ion-pairing, or ion exchange HPLC methods.

Results: MPO₂—(NHR⁶⁻) or metabolites thereof are not detected followingexposure of the prodrugs to carboxylesterase, alkaline phosphatase orplasma.

Example C Activation of Phosphoramidate Prodrugs by Rat Liver Microsomes

Methods: The microsomal fraction is prepared from fresh, saline-perfusedrat liver. Liver tissue is homogenized in three volumes (w/v) of 0.2 MKH₂PO₄ buffer pH 7.5, containing 2 mM MgCl₂ and 1 mM EGTA. Thehomogenate is centrifuged at 10,000 g for 1 hour and the supernatantrecovered. The supernatant fraction is then recentrifuged at 100,000 gto pellet the microsomal fraction. The pellet is resuspended inhomogenization buffer and recentrifuged. This process is repeated twiceto ensure complete removal of cytosolic enzyme activities. After thelast centrifugation, the microsomal pellet is resuspended inhomogenization buffer at a final protein concentration of about 14mg/ml. Reaction mixtures (0.5 ml) consist of 0.2 M KH₂PO₄ pH 7.5, 13 Mmglucose-6-phosphate, 2.2 mM NADP+, 1 unit of glucose-6-phosphatedehydrogenase, 0-2.5 mg/ml microsomal protein and 100 μM 7.1. Reactionsare carried out at 37° C. Aliquots are removed from the reactionmixtures at appropriate time points, and extracted with 60% methanol.The methanolic extracts are centrifuged at 14,000 rpm, and filtered (0.2μM) prior to analysis by HPLC. Standard HPLC techniques includingreverse phase, ion-pairing, or ion exchange chromatography are used.Eluted peaks are quantified relative to authentic standards of knownconcentration.

Alternatively, the activation of prodrugs is monitored by the depletionof NADPH, an essential cofactor in the reaction. This assay is performedin reactions mixtures consisting of 0.2 M KH₂PO₄, 0.22 mM NADPH, 0-2.5mg/ml microsomal protein, and 100 μM 28.4 or 30.1. Reactions aremonitored spectrophotometrically at 340 nm. A decrease in absorbance isindicative of cofactor depletion and thus of the enzymatic oxidation ofprodrug to MPO₂—(NHR⁶⁻).

Results: Prodrugs are converted to MPO₂—(NHR⁶⁻) in the presence, but notin the absence of, NADP+ (this cofactor is enzymatically reduced toNADPH by the dehydrogenase present in the reaction mixtures). Thisresult indicates that an oxidative step is involved in the activation ofthe prodrug. The rate of activation of prodrugs to MPO₂—(NHR⁶⁻) islinearly dependent on microsomal protein concentration, confirming thatactivation occurs by an enzyme-dependent mechanism.

Example D Activation of Phosphoramidate Prodrugs by Human Microsomes andthe Identification and Tissue Distribution of the Microsomal EnzymesInvolved in Activation

Methods: Human liver microsomes and cloned human P450 enzymesrepresenting all major isoforms are obtained from In Vitro Technologiesand Gentest Inc., respectively. Reaction mixtures typically contain 100mM potassium phosphate buffer, pH 7.4, 2 mM NADPH, 0-2 mg/ml microsomalprotein or cloned microsomal isozyme, and the prodrug at, for example, a100 μM concentration. Formation of MPO₂—(NHR⁶⁻) is monitored by standardreverse phase, ion pairing, or anion exchange HPLC methods. MPO₂(NHR⁶⁻)is detected by uv absorbance and quantified relative to authenticstandards. To determine the tissue distribution of the activatingenzyme, homogenates are prepared from key tissues including the liver,lung, intestine, kidney, heart, and skeletal muscle. The rate ofconversion of prodrug to MPO₂—(NHR⁶⁻) in each homogenate is assessed asdescribed for the microsomal reactions above. Alternatively, once themajor catalytic isoform is identified, immunohistological methods usingantibodies raised against the specific enzyme (Waziers et al 1989, J.Pharm. Exp. Ther. 254: 387), or molecular biological methods using PCRtechniques (Sumida et al 2000, BBRC 267: 756) are employed to determinethe tissue distribution of the activity.

Results: Prodrugs are transformed to MPO₂—(NHR⁶⁻) in the humanmicrome-catalyzed reaction at a readily measurable rate. In the screenof cloned microsomal isozymes, the CYP3A4 isozyme catalyzes activationof the prodrugs at the highest rate although other isozymes also showsome activity towards the prodrugs. The tissue distribution studiesindicate a high activity or abundance of the activating enzyme in theliver relative to other tissues.

Example E Identification of the P450 Isozyme Involved in PhosphoramidateProdrug Activation with Use of Specific Enzyme Inhibitors

Prodrugs are evaluated for human microsome-catalyzed conversion toMPO₂—(NHR⁶⁻) in the absence and presence of specific inhibitors of threemajor P450 isozymes: ketoconazole (CYP3A4), furafylline (CYPIA2), andsulfaphenazole CYP2C9).

Methods: Reactions (0.5 ml@37° C.) consist of 0.2 M KH₂PO₄, 13 mMglucose-6-phosphate, 2.2 mM NADP+, 1 unit of glucose-6-phosphatedehydrogenase, 0-2.5 mg/ml human microsomal protein (In VitroTechnologies, Inc.), 250 μM prodrug, and 0-100 μM P450 isozymeinhibitor. Reactions are stopped by addition of methanol to aconcentration of 60%, and filtered (0.2 μM filter). MPO₂—(NHR⁶⁻) isquantified by standard reverse phase, ion-pairing, or ion exchange HPLCmethods. Eluted peaks are quantified relative to authentic standards ofknown concentration.

Results: Ketoconazole inhibits the formation of MPO₂—(NHR⁶⁻) in adose-dependent fashion. The other inhibitors, furafylline andsulfaphenazole, show no significant inhibition. The results indicatethat CYP3A4 is the primary P450 isoform responsible for activationphosphoramidate prodrugs in human liver.

Example F Activation of Compound 1.3 by Recombinant CYP3A4

Activation of 1.3 was evaluated in reactions containing microsomes frombaculovirus-infected insect cells co-expressing human recombinant CYP3A4and cytochrome p450 reductase (Panvera Corp., Madison, Wis.).

Methods: Reaction mixture composition was similar to that described inExample D. Reactions were terminated by addition of methanol to a finalconcentration of 60% and products were analyzed by HPLC as described inExample A.

Results: 2-Amino-5-isobutyl-4-[2-(5-phosphonomonoamide)furanyl]thiazolewas generated from 1.3 at a rate of 4.2 nmoles/min./nmole CYP3A4.

Example G Activation of Antiviral Phosphoramidate Prodrugs in IsolatedRat Hepatocytes

The activation of 3.1 and 3.2 was evaluated by monitoring theintracellular generation of the corresponding nucleoside triphosphatesin rat hepatocytes.

Methods: Hepatocytes were prepared from fed, dexamethasone-inducedSprague-Dawley rats (300 g) according to the procedure of Berry andFriend (Berry, M. N., Friend, D. S. J. Cell Biol. 43, 506-520 (1969)) asmodified by Groen (Groen, A. K. et al. Eur J. Biochem 122, 87-93(1982)). Hepatocytes (62 mg wet weight/ml) were incubated in 2 ml Krebsbicarbonate buffer containing 10 mM glucose, and 1 mg/ml BSA.Incubations were carried out in a 95% oxygen, 5% carbon dioxideatmosphere in closed, 50-ml Falcon tubes submerged in a rapidly shakingwater bath (37° C.). 3.1 and 3.2 were dissolved in methanol to yield 25mM stock solutions, and then diluted into the cell suspension to yield afinal concentration of 250 μM. Two hours following administration,aliquots of the cell suspension were removed and spun through asilicon/mineral oil layer into 10% perchloric acid. The cell extracts inthe acid layers were neutralized by the addition of 0.3 volumes of 3MKOH/3M KHCO₃, and the extract was then centrifuged, filtered (0.2micron) and loaded onto an anion exchange HPLC column equilibrated with70% A (10 mM ammonium phosphate pH 3.5, 6% ETOH) and 30% B (1 M ammoniumphosphate pH 3.5, 6% ETOH). AraATP and acyclovir-triphosphate, theexpected metabolites of 3.1 and 3.2, respectively, were eluted from thecolumn with a linear gradient to 80% B in 20 minutes. Detection was byuv absorbance at 254 nm. Peak areas were quantified by comparison ofpeak areas to authentic standards spiked into naive hepatocyte extracts.

Results: Both prodrugs generated their respective triphosphates inisolated rat hepatocytes. Levels formed following two hours ofincubation are summarized below:

Intracellular concentration Compound nmoles/g 3.1 15.7 ± 16 3.2 13.3 ±3 

Example H Kinase Bypass

The generation of acyclovir triphosphate from a phosphoramidate prodrugof acyclovir (Compound 3.2) and acyclovir itself was compared in rathepatocytes.

Methods: 3.2 and acyclovir were evaluated in isolated rat hepatocytes at250 μM as described in Example G.

Results: Levels of acyclovir triphosphate formed following two hours ofincubation are summarized below:

Intracellular concentration Compound nmoles/g acyclovir <2 3.2 13.3 ± 3

The results demonstrate that a phosphoramidate prodrug of a parentnucleoside such as acyclovir, that is poorly phosphorylated in cells,can bypass the nucleoside phosphorylation step and thus generate higherintracellular levels of triphosphate than the free nucleoside.

Example I Inhibition of Glucose Production in Rat Hepatocytes byCompound 1.3

Compound 1.3 and its phosphonic acid parent compound,2-Amino-5-isobutyl-4-(2-furanyl)thiazole, were tested in isolated rathepatocytes for inhibition of glucose production.

Methods: Isolated rat hepatocytes were prepared from fasted SpragueDawley rats (250-300 g) as described in Example G. Hepatocytes (75 mgwet weight/ml) were incubated in 1 ml Krebs bicarbonate buffercontaining 10 mM glucose, and 1 mg/ml BSA. Incubations were carried outin a 95% oxygen, 5% carbon dioxide atmosphere in closed, 50-ml Falcontubes submerged in a rapidly shaking water bath (37° C.). After 10minutes of equilibration, lactate and pyruvate were added to 10 mM and 1mM concentrations, respectively. Addition of these gluconeogenicsubstrates was immediately followed by the addition of test compound toconcentrations ranging from 1-100 μM. After 1 hour, an aliquot (0.25 ml)was removed, and transferred to an Eppendorf tube and centrifuged. Theresulting supernatant (50 μA) was then assayed for glucose content usinga Glucose Oxidase kit (Sigma Chemical Co.) as per the manufacturer'sinstructions.

Results: 1.3 and 2-Amino-5-isobutyl-4-(2-furanyl)thiazole inhibitedglucose production in a dose dependent manner with IC50's of 8.5 and 2.5μM, respectively. Since 1.3 is not an inhibitor of FBPase in vitro,inhibition of gluconeogenesis in hepatocytes confirms that prodrug wasconverted to 2-Amino-5-isobutyl-4-(2-furanyl)thiazole intracellularly.The ˜3-fold lower potency of 1.3 relative to2-Amino-5-isobutyl-4-(2-furanyl)thiazole is consistent withtime-dependent activation of prodrug to parent compound in cells.

Example J Activation of Antidiabetic and Antiviral PhosphoramidateProdrugs in Induced Rat Hepatocytes

Compounds 1.7 and 12.1 was tested for activation in hepatocytes isolatedfrom rats in which CYP3A4 expression was induced by treatment withdexamethasone. This treatment results in more extensive intracellularactivation of phosphoramidate prodrugs.

Methods: Rats were treated with dexamethasone (50 mg/kg,intraperitoneally) for 4 days as described (Brain E G C et al 1998, Br.J. Cancer 7: 1768). Isolated hepatocytes were prepared, and prodrugstested for activation as described in Example G.

Results: 1.7 activated to yield 20.5±3 nmole/g levels of2-Amino-5-methylthio-4-[2-(5 phosphono)furanyl]thiazole following twohours of incubation. 12.1 generated 110±0.2 mmoles 1 g acyclovirtriphosphate.

Example K Cytotoxicity Testing of Phosphoramidate Prodrugs in Non-CYP3A4Expressing Cell Lines

Administration of a drug in a plasma-stable prodrug form can reducesystemic toxicities by limiting exposure to free MPO₂—(NHR⁶⁻) andmetabolites. This potential advantage of prodrug delivery isdemonstrated by comparing the toxicity profile of the prodrug and thecorresponding parent compound in a cell line which does not express theCYP3A4 enzyme required for prodrug activation.

Methods: The choice of cell line is dictated by the known toxicityprofile of the parent compound. If the toxicity profile of a parentcompound is unknown, a panel of different cultured cell lines can betested. Cells are exposed to a range of prodrug and parent compoundconcentrations for hours to days. Viability is the measured by Trypanblue exclusion, enzyme marker leakage, incorporation of labeledthymidine into DNA or other standard method.

Results: Phosphoramidate prodrugs are either noncytotoxic or cytotoxicat considerably higher concentrations than the corresponding parentcompound. The latter profile suggests that even if systemic exposure tothe prodrug is high, it is likely to show reduced peripheral toxicity invivo relative to free parent compound. Testing prodrugs in this mannerallows an assessment of the intrinsic toxicity of the prodrug and can beused to select prodrugs with favorable cellular safety profiles.

Example L Cytotoxicity Testing in Cellular Systems that CatalyzePhosphoramidate Prodrug Activation

Cytotoxicity testing in systems in which phosphoramidate prodrugs areactivated allows an assessment of the safety of MPO₂—(NHR⁶⁻) and othermetabolites of the prodrug or parent compound that are generated.Several cellular systems are useful for testing: (a) fresh primarycultures of hepatocytes of human or other origin in which CYP3A4expression is maintained with an appropriate inducer such as Rifampicin,(b) cell lines in to which CYP3A4 and the requisite reductase arecotransfected, (c) cell lines that are coincubated with primaryhepatocytes that express CYP3A4, (d) cell lines that are coincubatedwith CYP3A4 containing micromal fractions or a cloned CYP3A4 preparationin addition to a standard NADPH recycling system. Prodrugs are incubatedin these systems for hours or days. Cytotoxicity is assessed by standardmethods such as the Trypan blue exclusion technique, the leakage ofenzyme markers, the incorporation of labeled precursors into cellularDNA, etc.

Example M Byproduct Toxicity Testing, In Vitro

Activation of certain phosphoramidate prodrugs, in addition togenerating MPO₂—(NHR⁶⁻), also results in the formation of anelectrophilic byproduct capable of alkylating cellular constituents. Asthe prodrugs in question are activated primarily in the liver, primaryrat hepatocytes, known to contain both the microsomal enzymes requiredfor prodrug activation as well as critical detoxification mechanisms,are the cell type of choice for testing the potential toxicity of thebyproduct formed.

Methods: Isolated rat hepatocytes are prepared and incubated with testcompound as described in Example G. Acetaminophen (0-10 mM) is used as apositive control in these tests since its microsomal metabolism is alsoknown to generate an electrophilic metabolite in these cells (Smolarek TA et al 1989, Metabolism and Disposition 18: 659). This metabolite isdetoxified by addition to glutathione, a protective thiol present in mMconcentrations in liver cells. Incubations with acetaminophen andphosphoramidate prodrugs are for 4 hours. Cellular viability is assessedby lactate dehydrogenase or other liver enzyme leakage and/or by Trypanblue exclusion. Intracellular glutathione stores are also quantified asdescribed (Baker M A et al 1990, Anal. Biochem. 190: 360).

Results: Acetaminophen depleted intracellular glutatione levels by 90%at the highest dose tested (10 mM) but did not compromise cellularviability as measured by Trypan blue exclusion. The data suggest thatthe byproduct generated as a result of acetaminophen metabolism wasreadily detoxified in the hepatocytes, and that the cellular capacityfor detoxicification was not exceeded. Activation of phosphoramidateprodrugs does not cause byproduct-related toxicities in this hepatocytesystem.

Example N Byproduct Toxicity Testing In Vivo

Toxicity of the electrophile generated following microsomal activationof certain phosphoramidate prodrugs is tested in the mouse.

Methods: Mice are administered various doses of prodrug andacetaminophen (a positive control, see Example M) and seriallysacrificed at various time points following drug administration. Liversare removed and processed for glutathione analysis as described inExample M, and for drug analysis by standard reverse phase, ion-pairing,or ion exchange HPLC methods. Plasma samples are subjected to a standardchemistry panel in which markers of liver function are evaluated.

Results: Acetaminophen exposure (50, 250 and 500 mg/kg) resulted in adose-dependent reduction of hepatic glutathione. Liver toxicity in theform of liver enzyme elevations and the appearance of necrotic lesionswas evident only at the highest dose, which depleted hepatic glutathioneby >80%. This is in accordance with the literature; glutathione forms anadduct with the electrophilic acetaminophen metabolite formed, andprotects against liver injury until a threshold level of depletion isreached (Mitchell J R et al 1973, J. Pharm. and Exp. Ther 187: 211).Phosphoramidate prodrug administration also results in glutathionedepletion, indicating that the byproduct formed is efficientlydetoxified. Liver injury is apparent only at very high doses well abovethe projected therapeutic doses in man. The data also showdose-dependent accumulation of MPO₂—(NHR⁶⁻) and/or its metabolitesfollowing phosphoramidate prodrug administration.

Example O Enhanced Delivery of Drugs to the Liver in Animals Pretreatedwith CYP3A-Inducing Agents

Hepatic CYP3A enzymes are induced in rats by treatment withdexamethasone or other suitable agent. Enhanced expression of CYP3A willresult in a higher rate of prodrug activation and thus enhance thedistribution of MPO₂—(NHR⁶⁻) and/or its metabolites to this organ.

Methods: Rats are treated with dexamethasone (50 mg/kg,intraperitoneally) for 4 days as described (Brain E G C et al 1998, Br.J. Cancer 7: 1768). Other CYP-inducing agents such as phenobarbital orrifampicin may also be used. The induced animals are then administeredprodrug orally or sytemically and serially sacrificed at various timepoints. Livers are removed and homogenized in perchloric acid (10%).Following clarification by centrifugation and neutralization,MPO₂—(NHR⁶⁻) and/or its metabolites in the homogenates are quantified bystandard HPLC methods. A similar study is conducted in uninducedanimals.

Results: The AUC of MPO₂—(NHR⁶⁻) and/or its metabolites in livers ofinduced rats will exceed that of uninduced rats. Enhanced liver deliveryof MPO₂—(NHR⁶⁻) and/or its metabolites resulting from CYP3A induction isexpected to result in increased efficacy.

Example P Oral Bioavailability of Phosphoramidate Prodrugs

The oral bioavailability (OBAV) of phosphoramidate prodrugs is estimatedby comparison of the area under the curve (AUC) of MPO₂—(NHR⁶⁻) and/orits metabolites generated in liver following oral administration to thatgenerated following intravenous administration. Alternatively, OBAV isdetermined by means of a urinary excretion assay of MPO₂—(NHR⁶⁻) and/orits metabolites following prodrug administration.

Methods: Rats are dosed orally and systemically with a phosphoramidateprodrug in a suitable vehicle and serially sacrificed at appropriatetime point following drug administration. Liver and plasma samples areobtained, processed, and analyzed for MPO₂—(NHR⁶⁻) and/or itsmetabolites by standard reverse phase, ion-pairing, or ion exchange HPLCmethods. Alternatively, animals are placed in metabolic cages followingdrug administration, and urine is collected for 24-48 hours. Thequantity of MPO₂—(NHR⁶⁻) and/or its metabolites excreted into urine isthen determined by HPLC analysis. Oral bioavailability is calculatedeither by comparing the AUC of MPO₂—(NHR⁶⁻) and/or its metabolites inthe liver or the quantity in urine following oral and systemicadministration.

Results: The data indicate that phosphoramidate prodrugs are suitablefor the oral delivery of MPO₂—(NHR⁶⁻) and/or its metabolites.

Example Q Enhanced Liver Delivery and Reduced Systemic Exposure ofMPO₂—(NHR⁶⁻) and/or its Metabolites Following Administration ofPhosphoramidate Prodrugs

Method: Rats are dosed either orally or systemically with aphosphoramidate prodrug, MPO₂(NHR⁶⁻), and/or its metabolites. Animalsare serially sacrificed at appropriate time points following drugadministration. Liver and plasma samples are obtained, processed, andanalyzed for MPO₂ (NHR⁶⁻) and/or its metabolites by standard reversephase, ion-pairing, or ion exchange HPLC.

Results: Administration of phosphoramidate prodrugs results in thegeneration of higher levels of MPO₂—(NHR⁶⁻) and/or its metabolites inliver and their reduced concentrations in plasma than when freeMPO₂—(NHR⁶⁻) is administered.

Example R Reduced Renal Excretion of MPO₂—(NHR⁶⁻) and/or its MetabolitesFollowing Administration of Phosphoramidate Prodrugs

Methods: Phosphoramidate prodrugs and respective MPO₂—(NHR⁶⁻) areadministered systemically to rats. Rats are subsequently caged inmetabolic cages and urine collected over a 24-48 hour period.MPO₂—(NHR⁶⁻) and/or its metabolites are quantified in urine by means ofstandard reverse phase, ion-pairing, or ion exchange HPLC methods. Byrelating the amount of parent compound/metabolites excreted in urine tothe dose administered, the % renal clearance is calculated.

Results: Renal excretion of MPO₂—(NHR⁶⁻) and/or its metabolitesfollowing phosphoramidate prodrug administration is lower than whenparent compound is administered. This result indicates that the renalexposure and thus the renal toxicity associated with certain parentcompounds and/or their metabolites may be avoided by administration ofthe compound in phosphoramidate prodrug form.

Example S Sustained Liver Drug Levels

Methods: Rats are dosed orally and systemically with a phosphoramidateprodrug or the corresponding MPO₂—(NHR⁶⁻) and serially sacrificed atappropriate time point following drug administration. Liver samples areobtained, processed, and analyzed for MPO₂—(NHR⁶⁻) and/or itsmetabolites by standard reverse phase, ion-pairing, or ion exchangeHPLC. Liver half-lives are determined with the aid of WinNonLin 1.1software (Scientific Consulting, Inc.).

Results: The half-life of MPO₂—(NHR⁶⁻) and/or its metabolites in liveris longer when administered in phosphoramidate prodrug form than whenfree MPO₂—(NHR⁶⁻) is administered. This result is consistent with theprodrug serving as a MPO₂—(NHR⁶⁻) reservoir and, by virtue of aprolonged in vivo half-life, providing sustained release of MPO₂—(NHR⁶⁻)and/or its metabolites to the liver.

1. A compound of formula I:

wherein: V, W, and W′ are independently selected from the groupconsisting of —H, alkyl, aralkyl, alicyclic, aryl, substituted aryl,heteroaryl, substituted heteroaryl, 1-alkenyl, and 1-alkynyl; ortogether V and Z are connected via an additional 3-5 atoms to form acyclic group containing 5-7 ring atoms, optionally 1 heteroatom,substituted with hydroxy, acyloxy, alkoxycarbonyloxy, oraryloxycarbonyloxy attached to a carbon atom that is three atoms fromboth Y groups attached to the phosphorus; or together V and Z areconnected via an additional 3-5 atoms to form a cyclic group, optionallycontaining 1 heteroatom, said cyclic group is fused to an aryl group atthe beta and gamma position to the Y adjacent to V; together V and W areconnected via an additional 3 carbon atoms to form an optionallysubstituted cyclic group containing 6 carbon atoms and substituted withone substituent selected from the group consisting of hydroxy, acyloxy,alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy,attached to one of said additional carbon atoms that is three atoms froma Y attached to the phosphorus; together Z and W are connected via anadditional 3-5 atoms to form a cyclic group, optionally containing oneheteroatom, and V must be aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl; together W and W′ are connected via anadditional 2-5 atoms to form a cyclic group, optionally containing 0-2heteroatoms, and V must be aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl; Z is selected from the group consisting of—CHR²OH, —CHR²OC(O)R³, —CHR²OC(S)R³, —CHR²OC(S)OR³, —CHR²OC(O)SR³,—CHR²OCO₂R³, —OR², —SR², —CHR²N₃, —CH₂aryl, —CH(aryl)OH, —CH(CH═CR²₂)OH, —CH(C≡CR²)OH, —R², —NR² ₂, —OCOR³, —OCO₂R³, —SCOR³, —SCO₂R³,—NHCOR², —NHCO₂R³, —CH₂NHaryl, —(CH₂)_(p-)OR¹², and —(CH₂)_(p)—SR¹²; pis an integer 2 or 3; with the provisos that: a) V, Z, W, W′ are not all—H; and b) when Z is —R², then at least one of V, W, and W′ is not —H,alkyl, aralkyl, or alicyclic; R² is selected from the group consistingof R³ and —H; R³ is selected from the group consisting of alkyl, aryl,alicyclic, and aralkyl; R⁶ is selected from the group consisting of —H,and lower alkyl, acyloxyalkyl, alkoxycarbonyloxy alkyl and lower acyl;R¹² is selected from the group consisting of —H, and lower acyl; each Yis independently selected from the group consisting of —O—, —NR⁶— withthe proviso that at least one Y is —NR⁶—; M is an agent selected fromthe group that when attached to H, PO₃ ², P₂O₆ ³⁻, P₃O₉ ⁴⁻ orP(O)(NHR⁶)O⁻ is a biologically active agent, but is not an FBPaseinhibitor, and is attached to the phosphorus in formula I via a carbon,oxygen, sulfur or nitrogen atom of the M group; with the provisosthat: 1) M is not —NH(lower alkyl), —N(lower alkyl)₂, —NH(loweralkylhalide), —N(lower alkylhalide)₂, or —N(lower alkyl)(loweralkylhalide); and 2) R⁶ is not lower alkylhalide; and pharmaceuticallyacceptable salts thereof.
 2. The compounds of claim 1 wherein M is anagent selected from M-P(O)(NHR⁶)O⁻, M-PO₃ ²⁻, M-P₂O₆ ³⁻, and M-P₃O₉ ⁴⁻,such that M-P(O)(NHR⁶)O⁻, M-PO₃ ²⁻, M-P₂O₆ ³⁻, and M-P₃O₉ ⁴ is abiologically active agent selected from the group consisting of anantiviral, anticancer, antihyperlipidemic, antifibrotic, antiparasiticagents, metalloprotease inhibitor, and TS inhibitor.
 3. The compounds ofclaim 2 wherein M is an agent selected from M-H, such that M-H is abiologically active agent selected from the group consisting of LdC,LdT, araA, AZT, d4T, ddI, ddA, ddC, L-ddC, L-FddC, L-d4C, L-Fd4C, 3TC,ribavirin, penciclovir, 5-fluoro-2′-deoxyuridine, FIAU, FIAC, BHCG,2′R,5′S(−)-1-[2-(hydroxymethyl)oxathiolan-5-yl]cytosine,(−)-b-L-2′,3′-dideoxycytidine, (−)-b-L-2′,3′-dideoxy-5-fluorocytidine,FMAU, BvaraU, E-5-(2-bromovinyl)-2′-deoxyuridine, Cobucavir, TFT,5-propynyl-1-arabinosyluracil, CDG, DAPD, FDOC, d4C, DXG, FEAU, FLG,FLT, FTC, 5-yl-carbocyclic 2′-deoxyguanosine, Cytallene, Oxetanocin A,Oxetanocin G, Cyclobut A, Cyclobut G, fluorodeoxyuridine, dFdC, araC,bromodeoxyuridine, IDU, CdA, F-araA, 5-FdUMP, Coformycin,2′-deoxycoformycin, ACV, GCV, penciclovir, (R)-9-(3,4 dihydroxybutyl)guanine, and cytallene.
 4. The compounds of claim 1 wherein M isselected from an agent selected from M-PO₃ ²⁻, such that M-PO₃ ²⁻ is abiologically active agent selected from the group consisting of PMEA,PMEDAP, HPMPC, HPMPA, FPMPA, and PMPA.
 5. The compounds of claim 1wherein MP(O)(NH R⁶)O, MPO₃ ²⁻, MP₂O₆ ³⁻, or MP₃O₉ ⁴⁻ is useful for thetreatment of diseases of the liver or metabolic diseases where the liveris responsible for the overproduction of a biochemical end product. 6.The compounds of claim 5 wherein said disease of the liver or metabolicdisease is selected from the group consisting of hepatitis, cancer,fibrosis, malaria, gallstones, chronic cholecystalithiasis, diabetes,atherosclerosis, and obesity.
 7. The compounds of claim 5 wherein saidbiochemical end product is selected from the group consisting ofglucose, cholesterol, fatty acids, and triglycerides.
 8. The compoundsof claim 7 wherein MPO₃ ²⁻ or MP(O)(NHR⁶)O⁻ is an AMP activated proteinkinase activator.
 9. The compounds of claim 1 wherein Y is —O— locatedadjacent to the W′ and W groups.
 10. The compounds of claim 1 wherein Yis —O— located adjacent to the V group.
 11. The compounds of claim 1wherein both Y groups are —NR⁶—.
 12. The compounds of claim 1 wherein V,W, and W′ are independently selected from the group consisting of —H,alkyl, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl,substituted heteroaryl, 1-alkenyl, and 1-alkynyl; or together V and Ware connected via an additional 3 carbon atoms to form an optionallysubstituted cyclic group containing 6 carbon atoms and substituted withone substituent selected from the group consisting of hydroxy, acyloxy,alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy,attached to one of said additional carbon atoms that is three atoms froma Y attached to the phosphorus.
 13. The compounds of claim 12 wherein Vis selected from the group consisting of aryl, substituted aryl,heteroaryl, substituted heteroaryl; or together V and W are connectedvia an additional 3 carbon atoms to form a cyclic substituted groupcontaining 6 carbon atoms and mono-substituted with a substituentselected from the group consisting of hydroxyl, acyloxy,alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy attachedto one of said additional carbon atoms that is three atoms from an Yattached to the phosphorus.
 14. The compounds of claim 13 wherein V isselected from the group consisting of aryl, substituted aryl,heteroaryl, and substituted heteroaryl.
 15. The compounds of claim 14wherein Z, W, and W′ are H; and R⁶ is selected from the group consistingof —H, and lower alkyl.
 16. The compounds of claim 14 wherein V isselected from the group consisting of phenyl, and substituted phenyl.17. The compounds of claim 14 wherein V is selected from the groupconsisting of 3,5-dichlorophenyl, 3-bromo-4-fluorophenyl,3-chlorophenyl, 3-bromophenyl, 3,5-difluorophenyl, and 4-pyridyl. 18.The compounds of claim 13 wherein together V and W are connected via anadditional 3 carbon atoms to form an optionally substituted cyclic groupcontaining 6 carbon atoms and mono-substituted with one substituentselected from the group consisting of hydroxy, acyloxy,alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy attachedto one of said additional carbon atoms that is three atoms from an Yattached to the phosphorus.
 19. The compounds of claim 18 whereintogether V and W form a cyclic group selected from the group consistingof —CH₂—CH(OH)—CH₂—, —CH₂CH(OCOR³)—CH₂—, and —CH₂CH(OCO₂R³)—CH₂—. 20.The compounds of claim 12 wherein said compound is selected from acompound of formula VI:

wherein V is selected froth the group consisting of aryl, substitutedaryl, heteroaryl, and substituted heteroaryl.
 21. The compounds of claim12 wherein said compound is selected from a compound of formula VII:

wherein Z is selected from the group consisting of: —CHR²OH,—CHR²OC(O)R³, —CHR²OC(S)R³, —CHR²OCO₂R³, —CHR²OC(O)SR³, —CHR²OC(S)OR³,and —CH₂aryl.
 22. The compounds of claim 12 wherein said compound isselected from a compound of formula VIII:

wherein Z′ is selected from the group consisting of —OH, —OC(O)R³, —OCO₂R³, and —OC(O)S R³; D³ is —H; D⁴ is selected from the group consistingof —H, alkyl, —OH, and —OC(O)R³.
 23. The compounds of claim 12 whereinW′ and Z are —H, W and V are both the same aryl, substituted aryl,heteroaryl, or substituted heteroaryl, and both Y groups are the same—NR⁶—, such that the phosphonate moiety:

has a plane of symmetry through the phosphorus-oxygen double bond.
 24. Amethod of enhancing oral bioavailability of a parent drug byadministering to an animal a compound of claim
 1. 25. The method ofclaim 24 wherein said oral bioavailability is at least 10%.
 26. Themethod of claim 24 wherein said oral bioavailability is enhanced by 50%compared to the parent drug administered orally.